Anti-(retro)viral conjugates of saccharides and acetamidino or guanidino compounds

ABSTRACT

Conjugates of a saccharide and an acetamidino- or guanidino-compound, of the formula:                    
     wherein A is CH 3  or NH 2 ; X is a linear or branched C 1 -C 8  alkyl chain optionally containing hydroxy, amino and/or oxo groups; n is an integer from 1 to 6, and Sac is the residue of a mono-or oligo-saccharide, provided that when A is NH 2  and X is -(CH 2 ) 3 —CH(NH 2 )—C(=O)-, the monosaccharide residue is not substituted at the position 1, and n is an integer from 2 to 6 when Sac is the residue of an oligosaccharide, are useful as antiviral, particularly as antiretroviral, agents, and can be used either alone or together with other compounds used in AIDS treatment such as AZT and/or protease inhibitors, for the treatment of HIV-infection, AIDS and manifestations of AIDS such as Kaposi sarcoma. Particularly preferred compounds are aminoglycoside-arginine conjugates in which the saccharide is a natural aminoglycoside antibiotic such as kanamycin, gentamycin and neomycin that is conjugated to arginine residues.

REFERENCE TO RELATED APPLICATIONS

The present application is the national stage under 35 U.S.C. 371 ofinternational application PCT/IL99/00704, filed Dec. 28, 1999 whichdesignated the United States, and which international application waspublished under PCT Article 21 (2) in the English language.

FIELD OF THE INVENTION

The present invention relates to antiviral compounds, more particularlyto peptidomimetic conjugates of saccharides, such as aminoglycosideantibiotics, with acetamidino and guanidino compounds, and to antiviral,including antiretroviral, pharmaceutical compositions comprising them.

ABBREVIATIONS AND DEFINITION

HIV: human immunodeficiency virus; RT: reverse transcriptase; RNAse:ribonuclease; UC781: a non-nucleoside RT inhibitor; AZT: azidothymidine;KS: Kaposi sarcoma; AIDS: acquired immunodeficiency syndrome; Tat:trans-activator of transcription; TAR: trans-activation responsive RNAregion; LTR: long terminal repeat; P-TEFb: positive transcriptionelongation factor b; CDK9: cyclin-dependent kinase; ALX40-4C: D-argininenonapeptide; CGP64222: peptide peptoid mimetic of Tat basic domain;HeLa: human epithelial cell line derived from cervical cancer; CXCR4:CXC (α-chemotactic cytokines related to interleukin-8, containing C-X-Cmotif in their sequence, e.g. SDF-1α) chemokine receptor 4; CD4: clusterof differentiation 4 (characteristic receptor of T-helper cells); CCR5:CC (β-chemotactic cytokines, containing CC motif in their sequence)chemokine receptor 5; PBMC: peripheral blood mononuclear cells; T22:octadeca peptide, CXCR4 antagonist; AAC: aminoglycoside-arginineconjugates; R52: Tat-derived model undeca peptide, containing a singlearginine moiety at position 52 of native Tat protein, in the strand oflysines; R4K: tetra-argininamido kanamycin A conjugate; R3G:tri-argininamido gentamicin C conjugate; MMP: α-methyl D-mannopyranosideRMMP: mono-argininamido MMP conjugate; R4GC1a: tetra-argininamidogentamicin C1a isomer conjugate; GABA: γ-aminobutyric acid; GB4K:tetra-γ-(N-guanidino) butyramido-kanamycin A conjugate; NeoR:hexa-argininamido neomycin B conjugate; EIAV: equine infectious anemiavirus; ED: equine dermal fibroblasts; DMF: dimethyl formamide; DCC:dicyclohexyl carbodiimide; M.p.: melting point; Pd/C: palladium oncharcoal catalyst; TFA: trifluoro acetic acid; FABHRMS: fast atombombardment high resolution mass spectroscopy; HSQC: heteronuclearsingle-quantum coherence; TOCSY: total correlation spectroscopy; RRE:Rev responsive RNA element; CAT: chloramphenicol acetyl transferase;DTT: dithiotreitol; EDTA: ethylenediamine tetraacetic acid; CI₅₀:concentration of compound, that causes 50% inhibition of Tat-TARinteraction; CE₅₀: concentration of 50% elution from affinity column;CD₅₀: 50% binding concentration, related to K_(d); K_(d): dissociationconstant; LAN-1: human neurioblastoma cell line; MPC-11: murineplasmocytoma cell line; MT-2, MT-4: human T-lymphoma cell lines,transfected with HTLV-I; HTLV-I, HTLV-II: Human-T-lymphoma virus type Ior II; DMEM: Dulbecco modified Eagle's medium; FCS: fetal calf serum;polybrene: hexadimetrine bromide; pfu: plaque forming unit; ELISA:enzyme-linked immuno sorption assay; P4-CCR5 MAGI: human cell line ofmonocyte/macrophages origin; HUVEC: human umbilical vascular endothelialcells; SUP-T1: human T-cell line; cpe: cytopathic effect; IC₅₀: 50%inhibitory concentration; CC₅₀: 50% cytotoxic concentration; EC₅₀: 50%effective concentration; TI₅₀: 50% in vitro therapeutic index (ratioCC₅₀/EC₅₀); SDS: sodium dodecyl sulfate; PAGE: polyacrylamidegel-electrophoresis; TLC: thin layer chromatography; HRP: horseradishperoxidase; SDF-1α: stromal cell derived factor 1, subtype α, thenatural ligand of CXCR4; IL2: interleukin 2; IgG: immunoglobulin G; mAb:monoclonal antibody; 12G5: anti-CXCR4 mAb; 2D7: anti-CCR5 mAb; Leu3a:anti-CD4 mAb; PE: phycoerythrin; FITC: fluorescein isothiocyanate;RANTES: regulated on activation normal T-cell expressed and secretedchemokine; MPD: methyl pentandiol; SIR: single isomorphus replacement;SIRA: single anomalous replacement; MAD: multiple anomalous diffraction.

BACKGROUND OF THE INVENTION

The transactivation responsive RNA (TAR) region of humanimmunodeficiency virus (HIV) long terminal repeat (LTR) regulates theviral gene expression via interaction with the HIV transactivatorprotein, Tat, and thus is an attractive target for drug designstrategies (Gait and Karn, 1995). TAR is found at the 5′ end of allHIV-1 transcripts. It adopts a hairpin secondary structure consisting ofa highly conservative hexanucleotide loop and a three-nucleotide bulgeflanked by two double-stranded stems (Calnan et al., 1991 a, b). TAR isa positive enhancer that stimulates the synthesis of productivetranscripts. It is unique in terms of eukariotic transcription controlbecause it only functions as an RNA element. The activation by Tat isentirely dependent on the presence of the TAR RNA sequence. Tatactivates expression by specific binding to TAR, which increases viralmRNA production several hundred-fold by stimulation of the elongationcapacity of RNA polymerase II (Kingsman and Kingsman, 1996). HIV Tatbinds the cyclin T subunit of P-TEFb and recruits P-TEFb to the HIV-1LTR promoter. This process requires binding of Tat to the TAR bulge andof cyclin T to the TAR loop. The cyclin T associated CDK9 kinase theninduces phosphorylation of the C-terminal domain of RNA polymerase II,and of other polymerase II-associated proteins, leading to thetransition from non-processive to processive transcription (Cullen,1998).

Binding of Tat protein to TAR is mediated by the nine amino acid regionRKKRRQRRR (residues 49-57) of the protein (e.g. Calnan et al., 1991 a,b; Churcher et al., 1993). The nona-arginine peptide (R₉) binds to TARwith the same affinity and specificity as the above wild-type Tatpeptide, whereas the nona-lysine peptide (K₉) binds to TARnon-specifically and with a ten-fold lower affinity. The R₉-containingTat mutant protein gives wild-type trans-activation activity and is100-fold more active than the K₉-containing protein. Insertion of asingle arginine moiety at position 52 or 53 (synthetic peptides R52 ofthe sequence YKKKRKKKKA or R53) restores RNA-binding affinity andspecificity of the peptide as well as its trans-activation potency(Calnan et al., 1991 b). Mutagenesis studies on TAR RNA demonstratedthat the bulge (U23-C24-U25) and two base pairs at both sides of thebulge (e.g. Cordingley et al,. 1990; Roy et al, 1990; Delling et al.,1992) are important for Tat binding. Full length Tat protein binds TARwith only moderate affinity and specificity in vitro. The first 37N-terminal amino acids of the Tat protein decrease its affinity to TARin comparison to the specific recognition Tat (38-72) peptide (Rana andJeang, 1999). It was shown that human cyclin T1 promotes cooperativebinding of Tat protein to TAR RNA in vitro and mediates trans-activationin vivo. Although cyclin T1 does not bind TAR RNA, it may interact withthe TAR loop in a ternary complex of cyclin T1-Tat-TAR (Wei et al.,1998).

NMR structures of HIV TAR bound to different ligands, e.g. as peptidesthat mimic the basic region of Tat, arginine and arginineamide, showthat the ligands bind to the TAR RNA major groove (Puglisi et al., 1992,1993; Aboul-ela et al., 1995, 1996). The bulge structure allows ligandsto access the major groove of TAR, which induces folding in the bulgeand formation of unusual base-triples (Puglisi et al., 1992, 1993,Aboul-ela et al., 1995, 1996). The TAR RNA hairpin can adopt two majorconformations. In the absence of ligands, the bulge nucleotides stackwithin the RNA stem, severely distorting its helical continuity (Puglisiet al., 1992; Aboul-ela et al., 1996). When either L-arginineamide orthe Tat peptide bind to TAR, the bulge nucleotides loop out of theremaining stem, allowing the upper and the lower stem helices to stackcoaxially (Puglisi el al., 1992, 1993). The NMR structure ofL-arginineamide bound to TAR suggests proximity between the bulge andapical loop across the RNA major groove (Aboul-ela et al., 1995, 1996).The specific interactions between HIV Tat protein and TAR RNA are stillunknown but could be modeled as a basic ax-helix of Tat peptide lying inthe major groove of TAR (Mujeeb et al., 1996).

Tat-derived basic peptides, as well as the oligocationic peptide andpeptoid Tat mimetics bind TAR RNA with high affinity in vitro (e.g .Calnan et al., 1991 a, b; O'Brien et al., 1996; Hamy et al., 1997; Huqet al. 1997, 1999 a). Tat-mimetic compounds ALX40-4C (O'Brien et al.,1996) and CGP64222 (Hamy et al., 1997), that target TAR RNA, demonstratea pronounced antiviral activity. D-Tat peptide, derived from Tat 37-72sequence, binds to TAR RNA major groove and interferes with thetranscriptional activation by Tat protein in vitro and in HeLa cells(Huq et al., 1999 a).

The functions of Tat protein in viral progeny are not limited to HIVtrans-activation event. Tat trans-activates a number of cellular genesand acts as chemokine analogue, while secreted extracellularly (Albiniet al., 1998). Tat induces positive chemotaxis of human monocytes andmonocyte-derived dendritic cells (Benelli et al, 1998). ExtracellularTat protein was shown to up-regulate the expression of CXCR4 chemokinereceptor in primary-resting CD4+ T-cells (Secchiero et al., 1999) aswell as CCR5 receptor on monocytes (Weiss et al., 1999), which serve asco-receptors of viral entry for T-tropic and M-tropic HIV strains,respectively (Berger et al., 1999). It was shown, that HIV Tat protein,released by infected cells, differentially induces CXCR4 and CCR5expression in peripheral blood mononuclear cells (PBMC), and promotesinfectivity of both M- and T-tropic HIV-1 strains (Huang et al., 1999).The discovery of chemokine receptors as cofactors involved in the entryof HIV in the host cell has renewed the interest in the early steps ofvirus replication as a target for therapeutic intervention. A number ofcompounds have been described to interact with CCR5, the chemokinereceptor used by macrophage-tropic (MT, R5) strains of HIV (Simmons etal., 1997). Two other groups have also described newly identified CXCR4antagonists: ALX40-4C (Doranz et al., 1997) and T22 (Murakami et al.,1997), an octadecapeptide with 8 positive charges or its derivatives(Arakaki et al., 1999).

Another function of Tat protein (in synergism with inflammatorycytokines) is in induction of angiogenesis and the development of Kaposisarcoma in AIDS patients (Barillari et al., 1999). All the above Tatfunctions are dependent on the presence of the basic domain in theprotein structure. However, Tat has multiple domains, and two of themmediate the cellular and viral effects of extracellular Tat. Peptides,derived from cystein-rich domain and (possibly in combination with)basic domain were found to mimic the effects of a whole Tat protein inHIV-infected cells (Boykins et al., 1999).

Among natural RNA targeting molecules, aminoglycoside antibiotics haveinteresting properties that make them similar to peptide RNA binders.They are known to bind efficiently to RNA, such as 16S RNA or intronestype I (von Ahsen et al. 1992). Neomycin B and tobramycin inhibit HIVRev-RRE interaction in vitro at concentrations of 1-10 μM, whereaskanamycin and gentamicin do not display any inhibition at 100 μM. Recentexperiments have demonstrated that among the aminoglycoside antibiotics,neomycin has the greatest inhibitory effect on Tat binding to TAR invitro in the range of 1-100 μM. This phenomenon was attributed to thedirect association of the aminoglycoside antibiotic with TAR RNA in itslower stem (Wang et al., 1998).

SUMMARY OF THE INVENTION

It has now been found, in accordance with the present invention, that bycombining a carbohydrate skeleton, either a mono or an oligosaccharidesimilar to aminoglycoside antibiotics with side-chains of variablelength bearing a guanidine moiety or a chemical group with a similargeometry and/or charge properties resembling peptide side chains, a newclass of peptidomimetic TAR RNA binders is obtained that are anti-HIVcompounds and suppress viral replication (HIV-1 and EIAV) by inhibitingtransactivation by Tat as well as by blocking viral entry to cellsthrough chemokine-receptor-dependent mechanism. These relatively lowmolecular weight compounds, which mimic the functions of Tat proteinbasic domain, are the first examples of substances composed of acarbohydrate core conjugated to L-arginine or similar compounds withside chains, bearing guanidino or acetamidino groups that efficientlybind to TAR RNA as well as efficiently inhibit HIV-1 entry to T-cells.

The present invention thus relates to conjugates of saccharides andacetamidino or guanidino compounds of the formula I:

 wherein

A is CH₃ or NH_(2;) X is a linear or branched C₁-C₈ alkyl chainoptionally containing hydroxy, amino and/or oxo groups; n is an integerfrom 1 to 6, and Sac is the residue of a mono- or oligo-saccharide.

When A is CH₃, the conjugates are acetamidino saccharide conjugates, andwhen A is NH_(2,) the conjugates are guanidino saccharide conjugates.Sac may be the residue of a monosaccharide, in which case n is 1-5, orof an oligosaccharide, in which case n is 1-6.

Examples of monosaccharide conjugates according to the invention aremethyl 6-deoxy-6-(N-acetamidino)-α-D-mannopyranoside, methyl6-deoxy-6-guanidino-α-D-mannopyranoside and methyl6-deoxy-6-(N-L-argininamido)-α-D-mannopyranoside (the compounds 11, 10and 12 herein) of the formulas shown in Scheme 1 herein.

The natural or synthetic oligosaccharides are, for example, anaminoglycoside antibiotic such as, but not limited to, neomycin,kanamycin or gentamicin, or synthetic oligosaccharides. Theaminoglycoside-arginine conjugates of the invention will also sometimesbe designated herein in the specification as AAC.

The invention further provides pharmaceutical compositions comprising aconjugate of the invention and a pharmaceutically acceptable carrier,particularly for use as antiviral, more particularly as antiretroviral,compositions, alone or together with other anti-AIDS agents such as AZTor protease inhibitors.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C are schematic presentations of: (1A) Tat peptide (R52)binding to TAR RNA fragment (31 nucleotides) containing the sequence18-44 nt of HIV-1 LTR; (1B) Inhibition of Tat peptide (R52) binding tothe same TAR RNA fragment by the monosaccharide conjugate of theinvention methyl 6-deoxy- 6-(N-L-argininamido)-α-D-mannopyranoside(herein designated RMMP or compound 12); (1C) Binding oftetraargininamido-kanamycin A conjugate of the invention (hereindesignated R4K or compound 13) to TAR RNA.

FIG. 2 depicts the ¹H NMR TOCSY (total correlation spectroscopy) oftetraargininamido-kanamycin A conjugate R4K (compound 13) acquired at120 ms mixing time, on Bruker DPX 500 MHz in D₂O at 21° C. The spectrumreveals two sugar rings of kanamycin A, emphasized by a correlation withtheir anomeric protons (5.2 and 5.4 ppm). Additionally, one of the ringscorrelates with an AB system at 2.0-1.65 ppm (probably CH₂—OH). Areas ofstrong correlation at ˜1.6 and ˜3.28 correspond to arginine amidemoieties.

FIGS. 3A-3D present electrophoretic mobility shifts of the complexes ofTAR RNA with conjugates of the invention: (3A) Inhibition of Tat R52peptide binding to TAR RNA by conjugate RMMP (compound 12) (60 nM of TatR52, 6 nM TAR); (3B) Binding of conjugate R4K (compound 13) to TAR RNA(12 nM): a ratio of 2:1 R4K to TAR RNA in the complex is suggested; (3C)Binding of triargininamido gentamicin conjugate R3G (compound 14)mixture to TAR RNA (12 nM). Complexes of increasing molecular weight andprecipitation of RNA in the wells are observed; (3D) tetraargininamidogentamicin conjugate R4GC_(1a) (compound 15) binding to TAR RNA (20 nM):a ratio of 1:1 of R4GC_(1a) to TAR RNA in the complex is suggested. Theconcentration of Tat R52 in the control lanes is 200 nM.

FIGS. 4A-4C present footprinting studies of the binding sites ofaminoglycoside-arginine conjugates of the invention on TAR RNA.Approximately 100 nM 5′³²P end-labeled TAR was used per lane. Lane “OH”represents alkaline hydrolysis of TAR. Concentrations: Tat R52 peptide:2 to 4 μM (lanes 3, 4); R4K (compound 13): 10 to 20 μM (lanes 5, 6); R3G(compound 14): 4 to 8 μM (lanes 7, 8); GB4K (compound 19): 20 to 40 μM(lanes 9, 10). (4A) RNase A footprinting. Lane “ctrl” represents RNasecleavage in the absence of the binders. (4B) Uranyl photochleavage. Thereactions were performed in cacodylate buffer pH 6.5 (slightly acidicconditions modulate the uranyl cleavage, reflecting the conformationalchanges of nucleic acids). The samples were irradiated for 10 min at 420nm. Lane “ctrl” represents uranyl photocleavage in the absence of thebinders. (4C) Lead acetate footprinting. Lane “ctrl” represents leadacetate cleavage in the absence of the binders. The gels were analysedusing Storm 820 phosphoimager, quantitations were obtained usingImageQuant program.

FIG. 5 is a summary of the TAR RNA footprinting studies of FIG. 4. Theresults are derived from densitometry of the gels presented in FIG. 4.Bars represent % of protection calculated for each band compared to“control” (in the absence of binders) in each method. Negative peaksstand for enhancement of cleavage at the nucleotide (mainly by RNase A).Lead acetate results allow accurate assignment of the binding site,RNase A mainly probes secondary structure changes, uranyl nitrate, atour conditions at low pH, probes both binding site and conformationalchanges.

FIG. 6 is a schematic representation of Tat R52, R4K, R3G (compound 14)and GB4K binding sites on TAR RNA. The results indicate that more thanone molecule of R4K and R3G bind per one molecule of TAR. High affinitybinding site, similar to that of Tat R52, is located in the bulgeregion. Low affinity binding site is assigned to loop-stem junction.

FIGS. 7A-7E show the effect of yeast tRNA on TAR RNA band shifts andRNase A footprinting in the presence of Tat R52, R3G and R4K. 7A. Bandshifts of TAR RNA induced by binding of R4K and R3G (reproduced fromFIG. 3). 20 μl samples containing 10-12 nM ³²P-labeled TAR RNA and 1-5μM R4K or 0.5-4 μM R3G in 10 mM Tris-HCl (pH 7.5) buffer, containing 70mM NaCl, 0.2 mM EDTA and 5% glycerol, were incubated for 10 minutes at0° C. Following the incubation, the samples were analyzed byelectrophoresis on 10% native polyacrylamide gel (40:1). Gels were driedand visualized by autoradiography. R4K (left) forms 2:1 complexes withTAR RNA. R3G (right) forms complexes of increased molecular weight, andcaused precipitation of TAR RNA in the wells (not shown).

7B. Band shift of TAR complexes with R52, R4K and R3G in the presence ofan excess of yeast tRNA. 100 ng of yeast tRNA per lane do not inhibitthe binding of Tat R52, R4K and R3G to TAR RNA. Band shifts suggest onemolecule of aminoglycoside-arginine conjugate per one molecule of TARRNA in the complex. In the presence of 1 μg tRNA per sample, the bindingof both R52 and the conjugates was 90% inhibited.

7C. In the presence of 0.5 μg yeast tRNA, the RNase A footprinting ofTAR complexes with R52, R4K, R3G and GB4K has similar pattern as seen inFIG. 4. Binding of conjugates to low affinity binding site on TAR ispartially suppressed.

7D. Quantitation of band intensities of lane 5 of the gel, presented inFIG. 7C in comparison to lane 5 of the FIG. 4A.

7E. Schematic representation of conjugate binding to TAR RNA in thepresence of excess of tRNA.

FIGS. 8A-8B show the effect of R3G (8A) and R4K (8B) on equineinfectious anemia virus (EIAV) replication in equine dermal (ED)fibroblast cells. The cells were infected with 10 pfu EIAV per cell(superinfection) and were cultured in the medium, supplemented with³H-uridine. R3G and R4K were added to the medium in a range of 12.5 to100 μM. Viral titer was assayed every 3 days by radioactive uridineincorporation in the RNA of the viral particles. Each data pointrepresents averages of 6 (R3G) and 4 (R4K) experiments (variation within10%). R3G inhibited the viral growth at 12.5 and 25 μM, and R4K at 50and 100 μM.

FIGS. 8C-8D depict optical microscopy images of the cytopathic effect(cpe) development in EIAV-infected ED cells in the presence of R3G. Theimages were taken with Axiovert 100M (Zeiss) microscope using 40x planarobjective. C. After 13-15 days, EIAV-infected ED cells started to formsyncytia, that indicates the onset of EIAV cpe, D. The cells treatedwith 25 μM R3G preserved normal fibroblast phenotype at the same day ofthe infection.

FIGS. 9A-9B show cellular uptake of the aminoglycoside conjugate R4Kdetected by a fluorescent probe. (9A) Accumulation offluorescein-labeled conjugate R4K (R4K-fluorescein) probe in rathippocampal neurons, detected by confocal laser-scanning microscopy. Thecells were incubated in the presence of R4K-fluorescein (1 mg/ml) for 2hours; (9B) Laser scanning allows observation of intracellulardistribution of R4K-fluorescein in the neurons. Preferentiallyintranuclear accumulation of the probe is observed.

FIGS. 10A-10C show cellular uptake of the aminoglycoside conjugate R4Kdetected by a fluorescent probe. (10A) Light microscopy of humanperipheral blood mononuclear cells (PBMC); (10B) The same field as inFIG. 10A showing accumulation of R4K-FITC in PBMC, detected by confocallaser-scanning fluorescent microscopy. The cells were incubated in thepresence of R4K-FITC (1 mg/ml) for 1.5 hours; (10C) Superposition ofFIGS. 10A and 10B: fluorescence is highly detectable in the nuclei ofthe cells.

FIGS. 11A-11D present confocal microscopy images of live EIAV-infectedED cells stained with R3G-FITC. The images were taken with Axiovert 100M(Zeiss) microscope using 63x water immersion objective. (11A) Opticalmicroscopy of EIAV-infected ED cells, on day 16^(th). The cells aresignificantly damaged. (11B) The same fields, confocal fluorescentmicroscopy at 488 nm excitation. Fluorescent R3G derivative, R3G-FITC,accumulate in the cell nuclei. (11C) and (11D)—same as (11A) and (11B)at higher magnification (digital, 8x).

FIG. 12 is a schematic representation of binding sites of anaminoglycoside-arginine conjugate (AAC) of the invention on TAR RNA. TheNMR average structure of TAR RNA complex with arginine amide, wasobtained from Protein Data Bank, access code 1arj. Only nucleic acidchain is presented. Nucleotides highlighted in pink represent highaffinity binding site for AAC and Tat peptide. Binding of AAC to thissite is not inhibited by excess of tRNA. Nucleotides highlighted in bluerepresent low affinity binding site for AAC and aminoglycosides (e.gneomycin B). Binding of AAC to this site is inhibited by 10-fold excessof tRNA. Nucleotides highlighted in yellow represent sites of majorconformational changes induced by AAC binding to TAR. These positionsare cleaved by RNase A in TAR-AAC complex, but are not cleaved in theabsence of AAC. Small green balls represent proposed Mg⁺⁺and Ca⁺⁺bindingsites. Big yellow ball represents UO₂ ⁺⁺binding site.

FIGS. 13A-13B present antiviral activity of conjugates R3G and NeoR(compounds 14 and 20, respectively) against different dilutions of HIV-12D strain and clade C clinical isolate. Infection of MT2 cells wascarried out for 2 hrs with 1:1 to 1:16 dilutions of viral stock,followed by cell wash. Around 5×10⁴ cells were seeded per well in96-well plate and were incubated for 4 days with 20 μM R3G or 10 μMNeoR, until syncytia were observed. Cell viability was tested by thetetrazolium-based colorimetric method.

FIGS. 14A-14B show the effect of R3G and NeoR on HIV-1 clade C infectionof MT2 cells. Infection of MT2 cells was carried out for 2 hrs with orwithout 20 μM R3G or 10 μM NeoR followed by cell wash. Around 5×10⁴cells were seeded per well in 96-well plate and were incubated for 3days in the presence or absence of 10-20 μM R3G or 5-10 μM NeoR, untilsyncytia were observed (˜25% cpe). Cell viability was tested bytetrazolium-based colorimetric method.

FIGS. 15A-15B show the additive antiviral effect of R3G (15A) or NeoR(15B) and AZT on HIV-1 2D strain. Infection of MT2 cells was carried outfor 2 hrs followed by cell wash. Around 5×10⁴ cells were seeded per wellin 96-well plate and were incubated for 5 days with varyingconcentrations of R3G and AZT (15A) or NeoR and AZT (15B), untilsyncytia were observed. Cell viability was tested by tetrazolium-basedcolorimetric method.

FIGS. 16A-16B show flow cytometry analysis of the interaction of R3G orNeoR with the chemokine SDF-1α receptor CXCR4 on PHA-activated PBMC.FIG. 16A. PMBC lines ETH, derived from an HIV-negative Ethiopian bloodsample, and IS, derived from an HIV-negative Israeli blood sample(healthy controls) were incubated in RPMI 1640 containing 10% FCS in thepresence of PHA for 24 hrs. Cells (0.5×10⁶) were washed in ice-cold PBSand incubated for 30 minutes at 4° C. with monoclonal antibody (mAb)12G5 (anti-CXCR4), conjugated to phycoerythrin (PE) or with isotypecontrol mAbs in the presence or absence of R3G or NeoR. Then, the cellswere washed and fixed in 1% formaldehyde. For each sample 10,000 eventswere analysed in a FACScalibur™ System (Becton Dickinson). Data wereacquired and analysed with CellQuest™ software (Becton Dickinson). Inthe presence of 5 μM R3G or NeoR, the binding of 12G5 to PHA-activatedPBMC was suppressed. FIG. 16B. Effect of NeoR (5 μM) on binding of 12G5mAb to CXCR4 in PHA-activated PBMC. For each experiment cells wereincubated with isotype control mAb (a) or anti-CXCR4 mAb (12G5)conjugated to PE in the presence (b) or absence (c) of 5 μM NeoR. After30 min incubation at 4° C., cells were washed with PBS and analysed byflow cytometry.

FIG. 17 shows the effect of R3G (25 μg/ml) and R4K (25 μg/ml) on bindingof 2D7 mAb to CCR5, 12G5 mAb to CXCR4 and Leu3a mAb to CD4 instimulated-peripheral blood mononuclear cells. For each experiment,cells were incubated with mAbs 2D7, 12G5 or Leu3a conjugated to FITC, PEor PerCP, respectively, or isotype control mAb (dotted line), in thepresence (thick line) or absence (thin line) of 25 μ/ml of R3G (upperpanels) or R4K (lower panels). After 30 min incubation at 4° C., cellswere washed with PBS and analyzed by flow cytometry.

FIG. 18 shows that stromal cell-derived factor 1α (SDF-1α)-inducedsignaling via CXCR4 is blocked by R3G (upper panel) and R4K (lowerpanel). SUPT-1 cells were loaded with Fluo-3 fluorochrome and 10 secafter the first stimulation with the appropriate concentration of R3G orR4K, SDF-1α was given as a second stimulus at 20 ng/ml. Fluorescence wasmeasured in a Fluoroskan fluorimeter as described in Methods.

FIG. 19 shows that R3G and R4K inhibit binding of HIV-1 to CD4⁺cells.MT-4 cells were infected with 1×10⁵ pg of p24 antigen of HIV-1 (NL4-3)in the presence of different concentrations of the correspondingcompound. After 1 hour incubation a 37° C., cells were washed 3 times inPBS and p24 antigen bound to cells was determined by an ELISA test: R4K(black triangles), R3G (white triangles), dextran sulfate (blackdiamonds).

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, conjugates of saccharides andacetamidino or guanidino compounds are provided. These conjugatesdisplay high affinity to TAR RNA, being almost as efficient as Tat R52peptide.

The saccharide may be a simple monosaccharide such as a pentose, e.g.arabinose, xylose, ribose, or a hexose, e.g. glucose, mannose,galactose, fructose It may also be an oligosaccharide such as adisaccharide, e.g. sucrose, maltose, lactose, cellobiose, atrisaccharide, e.g mannotriose, raffinose, melezitose, or atetrasaccharide. Also encompassed within the definition of saccharideaccording to the invention are derivatives of saccharides such as, butnot limited to, glucosides, ethers, esters, acids and amino sugars.

The saccharide residue may be linked to the spacer X through anysuitable group, for example through an alkylene chain or, preferably,through an acylamino group. When the saccharide has no amino group, theamino group is introduced, for example at position 6 of themonosaccharide, by known methods such as by azide displacement on sugarsulphonates or halides, for example as described in Scheme 1 and Example2c herein for the preparation of compound 7.

In a preferred embodiment, the saccharide is a natural aminoglycosideantibiotic such as, but not limited to, kanamycin, gentamicin, neomycin,seldomycin, tobramycin, kasugamycin, etc.; or synthetic polyaminooligosaccharides. The aminoglycoside-arginine conjugates of theinvention are herein sometimes referred to as AAC. Preferred conjugatesaccording to the invention are those herein identified as R4K, R3G andNeoR.

The conjugates of the invention can be prepared by standard methods. Inone embodiment, the amino-sugar, e.g. aminoglycoside antibiotic, isreacted with arginine, thus obtaining conjugates of formula I wherein Ais NH₂ and X is —(CH₂)₃—CH(NH₂)—C(=O)—. In another embodiment, theconjugates can be prepared by reaction of α,ω-diamino acids of varyingchain length such as β-alanine, ornithine and lysine (2, 3 and 4methylene groups, respectively) or co-amino acids such as glycine(aminoacetic acid), β-amino propionic acid or γ-amino butyric acid, withthe aminoglycosides, and conversion of the terminal amino groups intoguanidino or N-acetamidino moieties by treatment with a variety ofguanilating agents such as O-methyl isourea, S-methyl isothiourea andothers or with O-ethyl acetimidate, according to known procedures.

The conjugates of the invention were found to be non-toxic for culturedcells, even at relatively high concentrations.

Based on peptide models of TAR RNA binding, NMR structures of TAR-ligandcomplexes and aminoglycoside-RNA interactions, we designed andsynthesized according to the present invention a set of novelpeptidomimetic substances, conjugates of aminoglycoside antibiotics witharginine. The combination of arginine residues and an aminoglycosidecore results in new compounds with structural features of both theoligocationic peptides and the aminoglycosides. Theseaminoglycoside-arginine conjugates (AAC) display high affinity to TARRNA in vitro: K_(d)′s of AAC-TAR complexes measured by gel-shifttechnique were found to be in the range of 20-400 nM, comparable to theK_(d) of the native Tat-TAR complex (6-12 nM). The finding thatgel-electrophoretic mobilities of the AAC-TAR differ from peptide-TARcomplexes, suggests that the stoichiometry of the complex between AACand TAR RNA in vitro is not equimolar. Their binding sites on TAR RNAwere assigned by RNase A, uranyl nitrate and lead acetate footprinting.The conjugates interact with TAR RNA in the widened major groove, formedby the UCU bulge and the neighbouring base pairs of the upper stemportion of TAR, the binding site of Tat protein and Tat-derived peptides(e.g. R52). Our results suggest an additional binding site of R4K andR3G compounds, in the lower stem-bulge region of TAR.

Aminoglycoside-arginine conjugates are non-toxic for cultured mammaliancells. The fluorescently labeled AAC of the invention efficientlyaccumulate in mammalian cell nuclei as was determined by confocalfluorescent microscopy studies. They are also expected to be resistantto enzymatic degradation that comprises one of the main problems forpeptide therapies. They further inhibit EIAV proliferation in culturedequine dermal a fibroblasts, as well as HIV-1 infection andproliferation in cultured human lymphocytes.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients. The carrier(s) mustbe acceptable in the sense that it is compatible with the otheringredients of the composition and are not deleterious to the recipientthereof.

The pharmaceutical composition will be administered by any suitablemethod including, but not being limited to, parenteral, e.g intravenous,intraperitoneal, intramuscular, subcutaneous, mucosal, e.g. oral,intranasal, intraocular.

For oral administration, the pharmaceutical preparation may be in liquidform, for example, solutions, syrups or suspensions, or in solid form astablets, capsules and the like. For administration by inhalation, thecompositions are conveniently delivered in the form of drops or aerosolsprays. For administration by injection, the formulations may bepresented in unit dosage form, e.g. in ampoules or in multidosecontainers with an added preservative.

The dose of the conjugate of the invention to be administered willdepend on the viral disease to be treated, on the individual's age andhealth condition, and other parameters as well known to physicians. Theconjugates may be used for the purpose of prevention and treatment ofHIV-infection and AIDS, either alone or in combination with othercompounds used in AIDS treatment such as, but not limited to, AZT and/orprotease inhibitors, as a part of anti-AIDS cocktails. The conjugates ofthe invention may be administered together with, before or after the AZTor protease inhibitors, in suitable administration forms and dosages asprescribed by the physicians. This combination therapy is suitable forthe treatment of HIV-infection, AIDS and AIDS conditions/manifestationssuch as Kaposi sarcoma.

The invention further relates to a method for treatment ofHIV-infection, AIDS or conditions/manifestations derived fromHIV-infection and AIDS, which comprises administering to an individualin need thereof, an effective amount of a conjugate of the invention,either alone or in combination with appropriate compounds used in AIDStreatment, to achieve alleviation of said infection or condition. Anexample of such condition is Kaposi sarcoma and examples of compoundsused in AIDS treatment are, without being limited to, AZT and proteaseinhibitors.

The invention will now be illustrated by the following non-limitingExamples.

EXAMPLES

For convenience and better understanding, the section of the Examples isdivided into two subsections: (I) the Chemical Section describing thesynthesis of the conjugates, and (II) the Biochemical and MolecularBiology Section describing the biological activity of the conjugates.

I CHEMICAL SECTION

In the Examples herein, the conjugates of the invention (10-20), othercompounds prepared for testing their suitability as antivirals (4,7-9)and the intermediates (1-3, 5, 6) will be presented by their respectiveArabic numbers in bold according to the following List of Compounds. Thecorresponding formulas appear in Scheme I (compounds 1-12), Scheme 2a(compound 13-R4K), Scheme 3a (compound 14-R3G), Scheme 3b (compound15-R4GC_(1a)), Scheme 4 (compounds 16-17), Scheme 4a (compound 20) andScheme 5 (compounds 18-19).

List of Compounds

1. Methyl α-D-mannopyranoside

2. Methyl 2,3,4-tribenzoyl-6-tosyl-α-D-mannopyranoside

3. Methyl 2,3,4-tribenzoyl-6cyano-α-D-mannopyranoside

4. Methyl 6-deoxy-6-cyano-α-D-mannopyranoside

5. Methyl 2,3,4tribenzoyl-6-azido-α-D-mannopyranoside

6. Methyl 6-deoxy-6-azido-α-D-mannopyranoside

7. Methyl 6-deoxy-6-amino-α-D-mannopyranoside

8. Methyl 6-deoxy-α-D-mannoheptopyranuronic acid amide

9. Methyl 6-deoxy-α-D-mannoheptopyranuronic acid amidoxime

10. Methyl 6-deoxy-6-guanidino-α-D-mannopyranoside

11. Methyl 6-deoxy-6-(N-acetamidino)-α-D-mannopyranoside

12. Methyl 6-deoxy6-(N-L-argininamido)-α-D-mannopyranoside (RMMP)

13. Tetraargininamido-kanamycin A conjugate (R4K)

14. Triargininamido-gentamicin conjugate (R3G)

15. Tetraargininamido-gentamicin (C_(1a)) conjugate (R4GC_(1a))

16. γ-(N-acetamidino)butyramido-neomycin B conjugate

17. γ-(N-guanidino)butyramido-neomycin B conjugate

18. Tetra-γ-(N-acetamidino)butyramido-kanamycin A conjugate

19. Tetra-γ-(N-guanidino)butyramido-kanamycin A conjugate (GB4K)

20. Hexa/penta-argininamido-neomycin B conjugate (NeoR)

EXAMPLE 1.

Preparation of compound 4

The synthesis of the cyano-monosaccharide 4 was carried out as depictedin Scheme 1 herein, by tosylation of the 6-hydroxy and benzoylation ofthe 2,3,4-hydroxy groups of the starting compound 1, conversion of the6-tosyl group of the obtained compound 2 into a 6-cyano group, andremoval of the benzoyl protecting groups from the obtained compound 3.

1a. Preparation of compound 2

To an ice-cold solution of 5 g methyl α-D-mannopyranoside (1) in 75 ccof pyridine, a cold solution of 5.5 g of p-toluenesulphonyl chloride in10 cc pyridine was added dropwise, and then the reaction mixture waskept at room temperature for 18 hrs, and the mixture was cooled again inan ice-bath, and 10 cc of benzoyl chloride was added. After anadditional 20 hrs at room temperature, the reaction mixture was pouredinto 1 liter of ice water, which resulted in the precipitation of pastymass. The aqueous solution was decanted, and the solid residue wastriturated with 100 cc of 2% sodium bicarbonate solution, filtered andwashed with water. The solid residue was refluxed with 50 cc of ethylalcohol for 5 min, filtered while hot, and the extraction was repeatedwith another 50 cc of ethyl alcohol. The remaining insoluble compound 2weighed 6.7 g after being dried over P₂O₅ overnight. M.p. 198° C.; ¹HNMR (CDCl₃): δ 7.3-8.25 (d and m, 19H, tosyl and benzoyl), 5.95—5.7 (mm3H, H₂₋₄), 5.015 (d, 1H, H₁), 4.38 (s 2H H_(6,6′)and m 1H H₅), 3.6 (s,3H, OMe), 2.45 (s, 3H, Me_(Ts)). [s=singlet, d=doublet, dd=doublet ofdoublets, AB=AB system, m=multiplet, mm=multiple multiplets, notresolved].

1b. Preparation of compound 3

Compound 2 (14.26 g), NaCN (5.5 g) and tetrabutylammonium bromide (3.5g) were dissolved in 100 cc of DMF. The reaction was allowed to proceedfor 40 hrs at room temperature, after which the reaction mass was pouredinto 2 liter of water and centrifuged. A pasty pellet was trituratedwith boiling ethyl alcohol and filtered while hot to remove unreactedcompound 2 (approximately 8%). Ethyl alcohol solution was decolorizedwith charcoal and evaporated in vacuo. Compound 3 was crystallized byaddition of methyl alcohol and recrystallized from acetone-methylalcohol, giving 4.8 g after drying over P₂O₅. M.p.144° C.; ¹H NMR(CDCl₃): δ 8.22, 8.07, 7.92 (d,d,d, 2H each, benzoyl), 7.38-7.66 (m, 9H,benzoyl), 6.0-5.8 (mm, 3H, H₂₋₄), 5.12 (d, 1H, H₁), 4.43 (m, 1H, H₅),3.70 (s, 3H, OMe), 2.90 (d, 2H, H_(6.6′)), ¹³C NMR (CDCl₃): δ 165.70,165.41, 165.32 (C=O benzoyl), 133.81, 133.68, 133.31, 129.93, 129.85,129.67, 128.68, 128.57, 128.32 (ring benzoyl), 116.38 (CN), 98.77 (C₁),70.12, 69.66, 69.34, 66.65 (C₂-C₅), 55.79 (Me), 21.52 (C₆).

1c. Preparation of compound4

5 g of compound 3 were added to 50 ml abs. MeOH containing 0.08 g MeONaand stirred for 16 hrs at room temperature. Sodium methoxide wasneutralized by addition of NH₄Cl (0.1 g), and the solution wasevaporated in vacuo. Resulting syrup was twice partitioned between 3 ccof water and 50 cc of benzene, decanting benzene and evaporating waterlayer to dryness. Resulting syrupy compound 4 was dissolved in 30 cc ofabs. acetone, filtered to remove salts, and evaporated in vacuo, giving,after drying over P₂0₅ in high vacuum, 1.95 g of pale yellow syrup. ¹HNMR (D₂O): δ 4.76 (d, 1H, H₁), 3.4-3.95 (mm, 4H, H₂₋₅), 2.9 (AB, 2H,H_(6.6′)) ¹³C NMR (D₂O): δ 101.11 (C₁) 69.98, 69.69, 69.35, 67.87(C₂₋₅), 54.76 (Me), 20.06 (C₆).

EXAMPLE 2.

Preparation of compound 7

The synthesis of the amino-monosaccharide 7 was carried out as depictedin Scheme 1 herein, by conversion of the 6-tosyl group of compound 2into a 6-azido group, removal of the benzoyl protecting groups from theobtained compound 5, and conversion of the 6-azido group of the obtainedcompound 6 into a 6-amino group.

2a. Preparation of compound 5

10 g of compound 2 and 3 g of NaN₃ were dissolved in 100 cc of DMF.Reaction was allowed to proceed for 24 hrs at 70° C. after which thereaction mass was poured into 1 liter of water. Compound 5, obtained asa solid, was recrystallized from acetone giving 8 g (quant.) afterdrying over P₂O₅. M.p. 111° C.; ¹H NMR (CDCl₃): δ 8.22, 8.07, 7.92(d,d,d, 2H each, benzoyl), 7.38-7.66 (m, 9H, benzoyl), 6.0—5.96 (m, 2H,H_(2,3)), 5.8 (m, 1H, H₄), 5.13 (d, 1H, H₁), 4.47 (m, 1H, H₅), 3.69 (s,3H, OMe), 3.62 (AB, 2H, H_(6.6′)).

2b. Preparation of compound 6

5.5 g of compound 5 were added to 50 ml abs. MeOH containing 0.08 gMeONa and stirred for 16 hrs at room temperature. Sodium methoxide wasneutralized by addition of NH₄Cl (0.1 g), and the solution wasevaporated in vacuo. Resulting syrup was twice partitioned between 3 ccof water and 50 cc of benzene, decanting benzene and evaporating waterlayer to dryness. Resulting syrupy compound 6 was dissolved in 30 cc ofabs. ethyl alcohol, filtered to remove salts and evaporated in vacuo,,giving, after drying over P₂O₅ in high vacuum, 1.3 g of transparentsyrup. ¹³C NMR (D₂O): δ 100.90 (C₁) 71.11. 70.18, 69.69, 67.30 (C₂₋₅),54.73 (Me), 50.92 (C₆).

2c. Preparation of compound 7

1 g of compound 6 was dissolved in 20 cc of ethyl alcohol. 0.5 g of 10%Pd on charcoal was added to the solution, and the mixture was stirredovernight under an atmospheric pressure of hydrogen. The catalyst wasremoved by centrifugation, and the ethanol solution was evaporated invacuo to yield, after drying over KOH, 0.85 g of title compound 7 ascolorless transparent glass. ¹H NMR (D₂O): δ 4.73 (d, 1H, H₁), 3.91,3.7, 3.55−3.51 (mm, 4H, H₂₋₅), 3.39 (s, 3H, OMe), 2.85 (AB, 2H,H_(6.6′)) ¹³C NMR (D₂O): δ 100.73 (C₁) 72.69, 70.33, 69.78, 68.09(C₂₋₅), 54.53 (Me), 41.36 (C₆).

EXAMPLE 3.

Preparation of compound 8

The synthesis of the amido-monosaccharide 8 was carried out as depictedin Scheme 1 herein, by conversion of the 6-cyano group of the compound 4of Example 1 above into a 6-acetamido group.

Thus, 300 mg of compound 4 were dissolved in 20 cc of ethyl alcohol with20 cc of 20% H₂ _(O) ₂, and 0.2 g of NaOH were added to the mixture.Reaction mixture was left overnight at 40° C. Resulting solution waspassed through Amberlite IRC-50 (H⁺) and Dowex 1×8 (OH⁻) ion exchangeresins, and concentrated to a thick syrup which was crystallized fromethyl alcohol/acetone, yielding 200 mg of compound 8 (dried overnight ina desiccator over P₂O₅). M.p. 177° C.; ¹H NMR (D₂): δ 4.67 (d, 1H, H₁),3.93, 3.73, 3.52 (mm, 4H, H₂₋₅), 3.37 (s, 3H, OMe), 2.63 (AB, 2H,H_(6.6′)) ¹³C NMR (D₂O): δ 176.27 (C_(amide)), 100.67 (C₁), 70.18,69.88, 69.78, 69.23 (C₂₋₅), 54.54 (Me), 37.44 (C₆). Structure confirmedby single-crystal X-ray diffraction.

EXAMPLE 4.

Preparation of compound 9

The synthesis of the amidoximo-monosaccharide 9 was carried out asdepicted in Scheme 1 herein, by conversion of the 6-cyano group of thecompound 4 of Example 1 above into a 6-acetamidoximo group.

Thus, 300 mg of compound 4 were added to 10 ml of abs. ethyl alcoholcontaining 0.3 g hydroxylamine base. Solution was heated to 70° C. and,after 30 hrs, refluxed for 10 more hrs. Resulting solution wascrystallized from abs. alcohol. Traces of hydroxylamine were removed bywashing crystals with 95% alcohol. After desiccation over KOH,crystalline compound 9 weighed 160 mg. It decomposes at 155° C. withoutmelting. ¹H NMR (D₂O): δ 4.69 (d, 1H, H₁), 3.90, 3.75−3.45 (mm, 4H,H₂₋₅), 3.35 (s, 3H, OMe), 2.50 (AB, 2H, H_(6.6′)) ¹³C NMR (D₂O): δ154.60 (C_(amidoxime)), 100.57 (C₁), 70.21, 69.99, 69.74, 69.58 (C₂₋₅),54.57 (Me), 32.08 (C₆). Structure confirmed by single-crystal X-raydiffraction.

EXAMPLE 5.

Preparation of compound 10

The synthesis of the guanidino-monosaccharide 10 was carried outaccording to Yoshimura et al., (1974), as depicted in Scheme 1 herein,by conversion of the 6-amino group of the compound 7 of Example 2 aboveinto a 6-guanidino group.

Thus, 630 mg of compound 7 and 620 mg S-methyl isothiourea sulfate weredissolved in 10 cc of 10% ammonia. The solution was left at roomtemperature for 4 days, then treated with Dowex 1×8 (OH⁻) and applied toa column of Amberlite IRC-50 (H⁺, 15 cc). The column was washedsuccessively with deionized water (200 cc), 5% aqueous ammonia (200 cc)and water (200 ml) and then eluted with 0.5M HCl. The neutral effluentwas collected until the pH changed, and it was evaporated to dryness.The dry residue was extracted with abs. ethyl alcohol, the extract wasevaporated, redissolved in water and then passed through Dowex 1×8(OH⁻). Upon evaporation, the dry syrupy title compound 10 weighed 385mg. ¹H NMR (D₂O): δ 4.74 (d, 1H, H₁), 3.92, 3.73−3.39 (dd, mm, 6H,H_(2-5,6.6′)), 3.37 (s, 3H, OMe), ¹³C NMR (D₂O): δ 158.97(C_(guanidine)), 102.16 (C₁) 72.24, 71.57, 71.08, 68.77 (C₂₋₅), 55.99(Me), 43.41 (C₆).

EXAMPLE 6.

Preparation of compound 11

The synthesis of the acetamidino-monosaccharide 11 was carried out asdepicted in Scheme 1 herein, by conversion of the 6-amino group of thecompound 7 of Example 2 above into a 6-acetamidino group.

Thus, 400 mg of compound 7 and 600 mg of ethyl acetimidate hydrochloride(Pinner, 1883) were dissolved in 10 cc of abs. alcohol with 1 cc oftriethylamine. The solution was left at room temperature for 6 hrs, thenevaporated. The residue was dissolved in 10 cc of water and treated withDowex 1×8 (OH⁻) and applied to a column of Amberlite IRC-50 (H⁺, 15 cc).The column was washed successively with deionized water (200 cc), 10%aqueous ammonia (200 cc) and water (200 ml) and then eluted with 0.5MHCl. The neutral effluent was collected until the pH changed, and it wasevaporated to dryness. The dry residue was extracted with abs. ethylalcohol, the extract was evaporated, redissolved in water and thenpassed through Dowex 1×8 (OH⁻). After evaporation, and overnight storageover KOH, a dry syrupy compound 11 weighing 150 mg was obtained. ¹H NMR(hydrochloride, D₂O): δ 4.74 (d, 1H, H₁), 3.96, 3.89−3.58 (m,mm, 1H, 5H,H_(2-5,6.6′)), 3.40 (s, 3H, OMe), 2.28 (s, 3H, CH_(3 amidine)) ¹³C NMR(D₂O): δ 165.84 (C_(amidine)), 100.79 (C₁) 70.05, 69.63, 69.61, 67.28(C₂₋₅), 54.64 (OMe), 42.58 (C₆), 18.25 (CH_(3 amidine)).

EXAMPLE 7.

Preparation of compound 12

The synthesis of the argininamido-monosaccharide 12 was carried out asdepicted in Scheme 1 herein, by conversion of the 6-amino group of thecompound 7 of Example 2 above into a 6-argininamido group.

Thus, 300 mg of compound 7 and 555 mg of L-Nα-carbobenzoxy, Nω-nitroarginine (Sigma Corp.) were dissolved in 4 cc of dry DMF. 324 mg ofN,N-dicyclohexyl carbodiimide (DCC) were added and the solution wasstirred at room temperature for 16 hours and then evaporated. Theresidue was washed with chloroform, dissolved in 15 cc of ethylalcohol/dioxane (1:1 v/v) and filtered. The solution was hydrogenated,using 0.3 g of 10% Pd/C, at atmospheric pressure for 8 hrs, and thenevaporated. The residue was dissolved in water and re-hydrogenated foradditional 24 hours using the same catalyst. The filtered solution waspassed through Dowex 1×8 (OH⁻) and then applied to a column of AmberliteIRC-50 (H⁺). The column was washed successively with deionized water, 2Naqueous ammonia and water and then eluted with 0.5M HCl. The neutraleffluent was collected until a pH change, and evaporated to dryness. Thedry residue was extracted with abs. ethyl alcohol, the extract wasevaporated, re-dissolved in water and then passed through Dowex 1×8(OH⁻). The solution was evaporated and the residue stored over KOHovernight, resulting in the dry syrupy title compound 12 which weighed140 mg. ¹H NMR (D₂O) contained characteristic protons of thecarbohydrate: δ 4.74 (H₁), 3.36 (OMe) and argininamide: δ 3.62(H_(αarg)), 3.19 (H_(βarg)), 3.62 (H_(γ-δarg)) ¹³C NMR (D₂O): δ 180.63(C_(amide)), 155.47 (C_(guanidine)) 103.70 (C₁) 73.45, 73.18, 72.69,70.96 (C₂₋₅), 57.53 (OMe), 57.22 (C_(αarg)) 43.60 (C₆), 42.737(C_(βarg)), 34.25 (C_(γart)), 27.32 (C_(δarg)). DEPT158 experiment hasconfirmed the nature of C₆ and C_(γ-δarg) peaks (CH₂).

EXAMPLE 8.

Preparation and characterization of the aminoglycoside-arginineconjugates 13-15, 20

8a. General procedure for preparation of conjugates 13-15, 20

Three general procedures were used for the preparation of the conjugatesof the invention 13-15 and 20 between arginine and the aminoglycosideantibiotics kanamycin, gentamicin and neomycin, which is alsoappropriate for other aminoglycoside antibiotics.

Method 1.

1-10 mmoles of aminoglycoside antibiotic are dissolved in 10 cc of dryDMF. For each amino group of the aminoglycoside, one equivalent of theprotected arginine derivative and 1.15 equivalent of DCC are added in4-5 portions during 8 hours. The reaction is allowed to proceed for 32hours at room temperature. The precipitated N,N-dicyclohexyl urea isremoved by filtration and the precipitate is washed with 2 cc DMF. Thefiltrate is then evaporated in vacuo, and washed with water andchloroform. The residue is dissolved in 20 cc. ethyl alcohol/dioxane(1:1 v/v), containing 1 equivalent of acetic acid per each positivelycharged group of the conjugate (based on theoretical yield) andhydrogenated at atmospheric pressure for 12 hours over 0.5 g 10% Pd/C.The solvents are evaporated and the residue is dissolved in 20 cc. waterand hydrogenation is continued for another 24 hours. The catalyst isremoved by centrifugation and the solution is evaporated in vacuo,resulting in faintly yellow, strongly basic viscous syrup, which isdissolved in 15 cc. water. The solution is then passed through a columnof Dowex 1×8 ion exchange resin (OH⁻form) to remove the free L-arginineand applied onto a column containing 20 cc. swollen Amberlite IRC-50 ionexchange resin (H⁺form). The column is washed successively withdeionized water, concentrated (5N) ammonia and water, and then theconjugate is eluted with 0.5M HCl. The neutral fractions are collectedand evaporated to dryness, and the solid residue is extracted withabsolute alcohol. Extracts are evaporated and redissolved in 15 cc. ofwater. Solution is passed through Dowex 1×8 ion exchange resin(OH⁻form), and evaporated to dryness, resulting in 100-300 mg of glassysolid of the desired conjugate of 85% -90% purity. Overall yield:10-15%. For higher purity, the crude conjugate was applied onto a 10×250mm HiBar column (C-18, Merck) and chromatographed in a 40-min gradientfrom 0 to 40% acetonitrile/0.1% trifluoroacetic acid (TFA) at 2.5ml/min. Peaks of interests were eluted at 22.5-27.5 min. The TFA saltsof the conjugates were used only for in vitro studies; free basesconjugates were utilized in the cell culture experiments, due tosignificant toxicity of the TFA anion. The conjugates were characterizedas described below.

Method 2.

1-10 mmoles of aminoglycoside antibiotic are dissolved in 3 cc ofdeionized water. For each amino group of the aminoglycoside, oneequivalent of the protected arginine derivative is added in 10 cc ofalcohol/dioxane/water mixture. The final composition of the solvent isaround 1:1:1 alcohol/dioxane/water that allows solubility of bothaminoglycoside and protected arginine. Equimolar quantity of awater-soluble carbodiimide (e.g. 1-ethyl, 3-(3-dimethyl aminopropyl)carbodiimide) is added in 4-6 portions during 12 hours at roomtemperature. The reaction progress is monitored by TLC onsilicagel-coated plates in a buffer containing isopropyl alcohol:25%water ammonia:chloroform in a ratio of 2:0.5:1. Typical duration of thereaction is 24 hours. The reaction mixture is then evaporated in vacuo,and washed with water. The residue is extracted with alcohol. Theremaining solid comprises 80-90% pure protected aminoglycoside-arginineconjugate (yield around 35%). It is hydrogenated and purified asdescribed in Method 1. Overall yield: 15-25%.

Method 3.

1-10 mM of aminoglycoside antibiotic are dissolved in 10 cc ofwater/alcohol/dioxane mixture. For each amino group of theaminoglycoside, one equivalent of N-hydroxy succinimide ester ofNα-tert-butoxy carbonyl, Nω-carbobenzoxy-L-ornitine is added during 12hours. The reaction mixture is evaporated, the residue is washed withdeionized water and the carbobenzoxy protective group is removed bycatalytic hydrogenation, as described in Method 1. The resultingconjugate of Nα-tert-butoxycarbonyl L-ornitine with the correspondingaminoglycoside antibiotic is treated with 12-15 equivalents of S-methylisothiourea sulfate in 5% aqueous ammonia for 3-5 days. The resultingconjugate of Nα-tert-butoxycarbonyl L-arginine with the correspondingaminoglycoside antibiotic is deprotected in 100% TFA in the presence of10 equivalents of dimethyl sulfide for 8 hours at room temperature. Thefurther purification and characterization of the conjugate are performedas in Method 1 (overall yield: 25-35%).

8b. General procedures for characterization of conjugates 13-15, 20

1 and 2D, ¹H and ¹³C NMR spectra of the compounds were taken in D₂O,(unless otherwise stated) at 21 ° C. using the following spectrometers:Bruker DPX 250 (250 MHz), Bruker DPX 400 (400 MHz) and Bruker DPX 500(500 MHz). Mass spectra of the compounds were obtained with FAB highresolution mass spectroscopy.

8c. Preparation/characterization of kanamycin A tetraarginine conjugate13 (R4K)

The starting antibiotic kanamycin (Fluka) was a mixture of three majorcomponents A, B and C (see Scheme 2), of which the A isomer was the mostabundant ˜80%). The conjugate of arginine with kanamycin was prepared bystandard peptide chemistry methods as described in Example 8a above. Asexpected, the kanamycin A derivative comprised approximately 90% of thearginine-kanamycin conjugate (R4K) mixture.

R4K was characterized as a free base and consisted, according to HPLC,of essentially one product, the kanamycin A derivative. The ¹H NMR (400MHz, D₂O) spectrum revealed the presence of the characteristic groups ofthe protons of arginine amide moieties at chemical shifts of (δ) 3.38(H_(α)), 3.21 (H_(β)) and 1.64 ppm (H_(γ,δ)) All the characteristickanamycin proton signals, in particular the anomeric hydrogens (asdoublets at 4.99 and 5.15 ppm), were observed. Integration afforded 1:4ratio of antibiotic to arginine components. A ¹³C NMR (100,609 MHz 10%D₂O) spectrum of R4K revealed carbon resonances of the C-arginine amidemoieties and the kanamycin moiety. The presence of antibiotic anomericcarbon signals at δ 101.77 and 101.07 ppm, the amide carbons at 181.13,180.81, 180.38, 179.76 ppm and the guanidino carbons at 159.47 ppm 5confirmed the structure of the aminoglycoside-arginine conjugate.FABHRMS of R4K revealed a mass peak of 1109.7 Da (calculated molecularweight of R4K: 1109.25 Da). A second peak of 1067.4 Da (−42.3 from masspeak) was attributed to a loss of aminoamino carbon fragment duringionization.

8d. 2D NMR studies of kanamycin A-tetra arginine conjugate R4K.

Since R4K consisted of essentially one substance, it was suitable for 2DNMR studies. Natural abundance [¹H; ¹³C] heteronuclear single-quantumcoherence (HSQC) and total correlation spectroscopy (TOCSY) spectra ofR4K were recorded on a Bruker DPX 500 MHz in D₂) at 21° C.

HSQC reveals the characteristic proton and carbon signal cross-peaks, inparticular from the two anomeric protons of the two saccharide rings (δ4.99 and 5.15 ppm) with corresponding carbons (δ101.1 and 101.8 ppm).TOCSY of R4K (FIG. 2) was acquired at 120 ms mixing time. The spectrumreveals two sugar rings of the antibiotic emphasized by correlation withtheir anomeric protons at 5.2 and 5.4 ppm. Correlations between thedeoxystreptamine axial and equatorial methylene protons (1.65 and 1.92ppm) and the ring system are observed. Methylene protons at C_(γ)andC_(δ)of arginine amide moieties (1.6-1.8 ppm) display correlation withthe protons at C_(β)(3.25-3.45) and it is possible to observe 4 arginineside chains (FIG. 3B.). Both spectra contributed much to the correctassignment of the R4K proton and carbon resonances.

8e. Preparation/characterization of gentamicin C₁ triarginine conjugate14 (R3G)

The starting antibiotic gentamicin C (Nova-Biochem) was a mixture of 3components C₁, C₂ and C_(1a), that differ by methylation of a singleamino group and adjacent CH₂ (see Scheme 3). The components wereseparated chromatographically, as previously described (Cooper et al.,1971). The ratio of the isomers was 4C₁: 3C₂: 1C_(1a). The preparationof the arginine conjugate with gentamicin C₁, R3G (Scheme 3a) wascarried out as described above in Example 8a.

R3G was characterized as an acetate salt (at pH 7.0). The substance is amixture of three products (as was proved by analytical HPLC), thederivatives of gentamicin C isomers (C₁, C2 and C_(1a)). Since thestructural difference between the three components is minor, thesubstance was used as a mixture in this study. Characteristic arginineamide ¹H NMR resonances of R3G were observed at ca 3.4 (H_(α)), 3.2(H_(β)) and 1.6 (H_(γδ)) ppm and characteristic antibiotic protonresonances, in particular the anomeric hydrogens, as three groups ofdoublets at 5.0-5.35; methyl singlets at 2.51 (N-Me of C₁, C₂ and C_(1a)isomers), 2.32 (N-Me of C₁), 1.21 (C-Me of C₁, C₂ and C_(1a)) ppm andmethyl multiplet at 1.04 (N-Me of C₁, C₂). Integration afforded 1:3average ratio of the gentamicin C components to arginine components,based on the composition of the antibiotic mixture used for thesynthesis. A ¹³C NMR spectrum revealed carbon resonances at severaldistinct regions characteristic of C-arginine amide moieties and threegentamicin isomeric moieties. Several C-amide carbon signals at179.8−179.3 ppm were observed as two groups of three peaks, as well asseveral minor peaks, indicating the presence of two major isomers, assuggested by the composition of the original antibiotic mixture.Guanidino carbon signals were detected at 160.04 ppm. FABHRMS of R3Grevealed two prominent mass peaks of 948.3 and 934.3 Da (calculated948.1 and 934.1 Da), which correspond to the triarginine derivatives ofC₁ and C₂ isomers, differing by a methyl group. Loss of the iminoaminocarbon fragment on chemical ionization by FABHRMS was also visible.

8f. Preparation and characterization of Gentamicin C_(1a) tetraarginineconjugate 15 (R4GC_(1a))

Preparation of the arginine conjugate of the pure C_(1a) isomer ofgentamicin, R4GC_(1a) (Scheme 3b) was carried out as described above inExample 8a. As expected, 4 arginine moieties were found in the conjugateas revealed by ¹H and ¹³C NMR spectroscopy.

8g. Preparation and characterization of hexa-argininamido-neomycin Bconjugate 20 (NeoR)

Preparation of the hexa/penta-arginine conjugate of neomycin B (seeScheme 4a) was carried out as described above in Example 8a. Argininemoieties and aminoglycoside parts were found in the conjugate asrevealed by ¹H and ¹³C NMR spectroscopy. Integration afforded average5.5:1 ratio of arginine to neomycin B parts, indicating the presence ofhexaarginine and pentaarginine derivatives of neomycin B in the mixture.

EXAMPLE 9.

Synthesis of γ-(N-acetamidino)butyric acid-Neomycin B conjugate(compound 16) and γ-(N-guanidino)butyric acid-Neomycin B conjugate (comp17)

Conjugates 16 and 17 were prepared by acetamidylation with ethylacetimidate or guanylation with O-methyl isourea or S-methyl isothioureaof the corresponding γ-amino butyric acid neomycin B conjugate, asdepicted in Scheme 4 herein.

9a. Preparation of the neomycin B conjugate with γ-amino butyric acid

The synthesis was carried out via the active ester of γ-amino butyricacid as depicted in Scheme 4, as follows:

2.2 g of N-hydroxysuccinimide ester of (Nγ-carbobenzoxy) γ-amino butyricacid, dissolved in additional 4 ml of formamide was added to 6 ml ofneomycin B base (500 mg) solution in formamide. The mixture was stirredfor 48 h at room temperature and then poured into 100 ml of 5% NaHCO₃solution, centrifuged and the syrupy pellet washed with 100 ml of water,suspended in chloroform and acetone (20 ml each) to extract unreactedmaterial. The pellet was dried and then dissolved in 20 ml of alcoholcontaining 0.7 ml of glacial acetic acid. The solution was hydrogenatedat atmospheric pressure over 10% Pd/C for 24 h. After removing thecatalyst by centrifugation and evaporation in vacuo the conjugate wasdeionized with Dowex 1×8 (OH form), absorbed on Amberlite IRC-50(H⁺form) and eluted from the Amberlite with 25% NH₄OH; 200-500 mg(20%-50% yield) of the conjugate was obtained after concentration of theeluate in vacuo. Methanol (abs.) can be substituted for formamide,especially in the case of kanamycin conjugate preparation.

Alternatively, synthesis may be accomplished using the standard DCCcoupling protocol, as described above.

9b. Preparation of conjugate 16

For acetamidylation, the neomycin B/γ-amino butyric acid conjugateobtained in Example 9a above is treated with O-ethyl acetimidate inabsolute ethanol for 1-2 days, resulting in acetimidylation of theterminal amino groups. The product is purified by ion exchangechromatography, yielding neomycin B/N-acetamidino butyric acid conjugate16.

9c. Preparation of conjugate 17

For guanylation, the neomycin B/γ-amino butyric acid conjugate obtainedin Example 9a above is treated with O-methyl isourea or S-methylisothiourea in water at basic pH for 34 days, resulting in guanylationof the terminal amino groups. The product is purified by ion exchangechromatography, yielding the neomycin B/γ-guanidino butyric acidconjugate 17.

EXAMPLE 10.

Synthesis of tetra-γ-(N-acetamidino)butyric acid-kanamycin A conjugate(compound 18) and tetra-γ-(N-guanidino)butyric acid-kanamycin A 10conjugate (compound 19, GB4K)

Conjugates 18 and 19 were prepared by acetamidylation with ethylacetimidate or guanylation with O-methyl isourea or S-methyl isothioureaof the corresponding γ-amino butyric acid kanamycin A conjugate (seeScheme 5) as described in Example 9 above with some modification. It wasnot necessary to purify the intermediate GABA-kanamycin conjugate. Bestresults were obtained when methanol was used as a solvent for theacylation of the kanamycin amino groups by succinimide ester of(N-carbobenzoxy) γ-amino butyric acid.

GB4K was characterized as an acetate salt. Characteristic ¹H signals ofguanidino butyric acid amide chains were observed as multiplets at 3.19(H_(β)), 2.32 (HO) and 1.86 (H_(γ,δ)) ppm. All characteristic signals ofthe kanamycin moiety were found, in particular the anomeric protons assinglets at 5.32 and 5.11 ppm. Integration afforded 1:4 ratio of theantibiotic to guanidino butyric acid amide parts. A ¹³C NMR spectrumrevealed characteristic groups of signals of both the guanidino-butyricacid amide and the antibiotic moiety. In particular, the antibioticanomeric carbon signals were observed at 100.03 and 98.04 ppm; the amidecarbons at 176.63, 176.22, 175.32 and 174.94 ppm and the guanidinocarbons at 157.33 ppm. Using FABHRMS the mass peak was found to be 993.5Da (calculated 993.1 Da). Loss of the iminoamino carbon fragment uponionization was also observed.

II BIOLOGICAL SECTION

Materials and Methods

(i) TAR RNA and Tat peptide preparation

The model Tat peptide (R52: YKKKRKKKKKA) was prepared by the WeizmannInstitute Chemical Services and was purified by reverse phase HPLC(C-18) (Lapidot et al., 1995). A 31-nt TAR RNA fragment (5′-GGC CAG AUCUGA GCC UGG GAG CUC UCU GGC C-3′) containing the sequence 18-44 of theHIV LTR (Aboul-Ela et al., 1995) was transcribed in vitro by T7 RNApolymerase (Promega) from a synthetic single strand DNA templatecontaining the 17 base double-stranded T7 promoter (both DNAoligonucleotides were prepared by the Weizmann Institute ChemicalServices). The RNA was transcriptionally labeled with α-³²P UTP,purified on a 12% polyacrylamide/7M urea gel (19:1acrylamide:bis-acrylamide) and was eluted from the gel by 0.5M ammoniumacetate, 10 mM magnesium acetate, 1 mM EDTA and 0.1% SDS. The sample wassubjected to phenol extraction and the RNA was precipitated from 70%ethanol. Purified RNA was dissolved in diethyl pyrocarbonate-treatedwater (DEPC water), its concentration was determined by UV absorption at260 nm and the specific radioactivity was determined by scintillationcounting on a LS 1701 “Bruker” counter.

Alternatively, chemically synthesized TAR RNA oligonucleotide(Dharmacon) was 5′-end labeled with 1 μl (γ-³²P) ATP (6000 Ci/mmol,Amersham) per 1 nmol of RNA using T4 polynucleotide kinase (Promega) ina buffer containing 70 mM Tris-HCl pH 7.5, 10 mM MgCl₂ and 5 mM DTT. Thelabeled RNA was successively extracted with water-saturated phenol,phenol-chloroform (1:1) and chloroform, and precipitated from 75%ethanol. All RNA samples were annealed by heating them to 95° C. for 5min, followed by slow cooling to room temperature in a buffer,containing 10 mM sodium cacodylate (pH 6.5) and 50 mM KCl. RNA puritywas analyzed by denaturing gel-electrophoresis. The alkaline cleavage ofTAR RNA was performed by incubation of 10 μl RNA samples (containing50-100 ng of 5′ ³²P labeled TAR RNA) with 10 mM NaOH for 3-5 min at roomtemperature. The reaction was stopped by addition of 1 μl 150 mM aceticacid.

(ii) Gel-shift assays

To study the affinity of the test compounds to TAR, gel-shift andgel-shift inhibition experiments were performed. The binding reactionmixtures (20 μl) contained ³²P-labeled TAR (18-44) RNA oligonucleotide(3-6 nM) and Tat R52 peptide (35-75 nM) in a binding buffer (10 mMTris-HCl pH 7.5, 70 mM NaCl, 0.2 mM EDTA and 5% glycerol). The reactionmixtures were incubated for 10 minutes on ice and resolved byelectrophoresis on a 10% non-denaturing polyacrylamide gel (40:1acrylamide:bis-acrylamide) at 200V, for 3 h at 4° C. Gels were dried andvisualized by autoradiography. Quantitations were obtained by opticaldensitometry of films. Alternatively, results were visualized byexposing the wet gels to Fuji phosphoimager plates, which were read on a“Storm 820” (Molecular Dynamics) phosphoimager. Different concentrationsof TAR RNA (6,12 and 20 nM) were titrated with various concentrations ofTat R52 in the binding reactions. CD₅₀ values were defined as the Tatpeptide concentration that displayed 50% binding to TAR RNA.

Binding inhibition by the monosaccharide derivatives was measured byadding several concentrations of each compound to reaction mixturescontaining 60 nM Tat 52 and 6 nM ³²P TAR, attaining 100% binding of TatTAR in the absence of inhibitors (Lapidot et al., 1995). The bindinginhibition values, CI₅₀, were defined as inhibitor concentrations thatdisplayed 50% inhibition of Tat TAR binding.

Binding of aminoglycoside-arginine conjugates (AAC) to TAR RNA wasmeasured by adding varying concentrations of each conjugate to reactionmixtures (20 μl) which contained 12 nM ³²P-labeled TAR RNA. Thereactions were analyzed as described above. CD₅₀ values were defined asthe conjugate concentrations that displayed 50% binding to TAR RNA.

(iii) Affinity chromatography on L-arginine-Amberlite column

Experiments with L-arginine-Amberlite affinity column were performed toobtain further evidence of the specificity of the conjugates binding toTAR (Geiger et al., 1996). The L-arginine-Amberlite resin was preparedas follows: Carboxyl groups of Amberlite IRC-50 resin were covalentlymodified with ethanolamine using 1-(3-dimethylaminopropyl)3-ethylcarbodiimide, a water-soluble coupler. The hydroxy ethyleneamideresin obtained was washed and dried, and L-arginine (12-16 μmols per mlof resin) was coupled to the hydroxy groups of the resin via an isoureamoiety using a standard cyanogen bromide protocol. The finalconcentration of arginine residues was 12-16 μmol per ml of resin. Theswollen L-arginino-Amberlite resin (200 μl) was packed into a plasticmini-column and equilibrated with buffer containing 40 mM Tris HCl, pH7.5, 250 mM NaCl, 0.5 mM EDTA and 5 mM MgCl₂ (buffer A). Approximately 1ng of ³²P-UTP labeled TAR RNA in buffer A was loaded on the column. Thecolumn was eluted 3 to 5 times with 400 μl portions of buffer A, untilno radioactivity was detected in the eluent. The total radioactivity inthe eluents did not exceed 3% of the total radioactivity loaded. Each ofthe test compounds was dissolved in buffer A and passed through thecolumn in 300 μl portions. Radioactivity of the eluates was determinedby scintillation counting on a LS 1701 “Bruker” counter. CE₅₀ values,the concentrations of inhibitors eluting 50% of the radioactive materialfrom the column, were determined. The eluate samples were proven tocontain labeled TAR RNA by electrophoresis on a denaturing 12%polyacrylamide/7M urea gel, followed by autoradiography.

The L-arginine-Amberlite affinity resin described above was found to bemore mechanically stable and of better affinity properties than theL-arginine-agarose resin previously used (Tao and Frankel., 1992). Thismay be due to the higher arginine concentration and 6-atom spacerbetween the polymer and the arginine residues.

(iv) RNase A footprinting.

In a typical experiment 10 μl of 5 mM cacodylate buffer pH 6.5,containing 25 mM KCl and 50-100 ng 5′ ³²P-labeled TAR RNA (approximately0.5-1 μM) were incubated with 200 pM RNase A (Sigma) at room temperaturefor 10 minutes in the presence of Tat R52 and the conjugates of theinvention at various concentrations. In the competition experiments, themixture was supplemented with 0.5 μg yeast tRNA (Sigma). After theincubation, 10 μl of formamide/bromphenol blue loading buffer was addedto the samples. The samples were heated to 80° C. for 2 min and wereresolved by electrophoresis on 40 cm×0.8 mm 20% polyacrylamidedenaturing gel (7M urea) for 3 hours at 55° C. The results werevisualized by a phosphoimager as described above and quantitated usingImageQuant software.

(v) Lead acetate footprinting.

In a typical experiment, 10 μl of 50 mM cacodylate buffer pH 6.5,containing 100 mM KCl, 1 mM MgCl₂ and 0.5 μg yeast tRNA were incubatedwith 50 ng 5′ ³²P labeled TAR RNA (approximately 0.5 μM) in the presenceof varying concentrations of Tat R52 and the AAC of the invention atroom temperature for 5 minutes. Cleavage reactions were initiated byaddition of Pb(OAc)₂ to final concentrations of 0.1 mM. After 20 minincubation, 10 μl of 90% formamide/bromphenol blue loading buffer,containing 100 mM EDTA, was added to the samples. The samples wereheated to 80° C for 2 min and were resolved by electrophoresis andquantitated as described above.

(vi) Uranyl nitrate photocleavage.

The procedure for photoinduced cleavage of TAR RNA by uranyl cation wasas following: 50 μM uranyl nitrate were added to 10 μl of 5 mMcacodylate buffer pH 6.5 or 5 mM KMOPS buffer, pH 7.5, containing 25 mMKCL and 1 μM 5′ ³²P labeled TAR RNA. Inhibitors or divalent metal saltswere added to the mixture at various concentrations. Tracing of thehypersensitive cleavage sites was performed in the presence of 37.5 μMcitrate. The samples were irradiated for 10-20 minutes at roomtemperature under a Philips T1 40 W/03 fluorescent tube, emitting lightat wavelength of 420 nm. Following irradiation, the samples weretreated, resolved by electrophoresis and quantitated as described above.

(vii) Cytotoxicity measurements

All the conjugates of the invention that were found to inhibit Tat-TARinteraction or to bind TAR RNA were tested for toxicity in the followingcell cultures: LAN 1 (human neuroblastoma), MPC 11 (murineplasmacytoma), MT4 (human T-lymphocytes) and ED (equine dermalfibroblasts). Approximately 2.5×10⁴ cells were seeded per each well ofthe standard 96-well “Falcon” tissue culture plate in 200 μl DMEMcontaining 10% heat-inactivated fetal calf serum (FCS). Differentconcentrations of the compounds were added to the wells in triplicates.After 24 to 48 hours incubation the cells were washed and the medium waschanged for the same amount of DMEM/10% FCS containing 0.5 μCi of³H-thymidine per well for 1 hour. Then the medium was removed, the cellswere washed with saline and the DNA was precipitated by ice-cold 10%trichloroacetic acid. The precipitate was solubilized in 50 μl of INNaOH, mixed with 4 ml of Ultima Gold® scintillation liquid (Packard) andcounted on a LS 1701 “Bruker” scintillation counter.

(viii) Cellular uptake using fluorescent probes

Uptake of the conjugate compounds by cells and intracellulardistribution was studied using fluorescent probes (Wells and Johnson,1994). The fluorescent derivative of the aminoglycoside-arginineconjugate (R4K=13) was prepared by reacting the conjugate 13 withfluorescein isothiocyanate (FITC, “Sigma”) in water-methanol-dimethylsulfoxide mixture for 24 hours. FITC was added in 1:1 molar ratio to theconjugate, which enabled only one random amino group of the conjugate toreact with FITC. The fluorescein-labelled conjugate was purified byextraction with acetone and absolute alcohol.

Hippocampal neurons from rat puppies were grown over rat glia cells onpolylysine-coated glass cover slides. The cells were incubated inHEPES-buffered saline containing 1 mg/ml of R4K-fluorescein probe for 2hours. The slides were washed several times with saline and studied byconfocal laser-scanning microscopy on “Axiovert 100M” (“Zeiss”)microscope, using excitation at 488 nm (argon-ion laser) and emissiondetected at 505-550 nm. The intense fluorescence, observed in the cells,indicated an efficient uptake of the probe; even higher fluorescence wasdetected in the cell nuclei (FIG. 4), suggesting that theR4K-fluorescein is associated with nucleic acids, as was expected. Theuptake of R4K into peripheral blood mononuclear cells (PBMC) was alsostudied, using the same technique. Human PBMC were separated from freshblood sample on Ficoll gradient by standard procedure. They were grownon polylysine-coated glass cover slides in RPMI medium, containing 10%fetal calf serum, for 24 hours. The cells were treated as above,R4K-fluorescein concentration was 0.1 mg/ml. As was expected, thefluorescent probe was observed in the nuclei in almost all cells (FIG.5), indicating binding to nucleic acids.

Equine infectious anemia virus (EIAV)-infected equine dermal fibroblasts(ED) at the late stage of infection were plated on collagen-coated glassslides and incubated in HEPES-buffered saline in the presence of 10μg/ml of FITC-labeled compounds (R4K and R3G) for 0.5-1 hour. The slideswere washed several times with saline and studied by confocallaser-scanning microscopy on the Axiovert 100M (Zeiss) microscope, using488 nm excitation (argon-ion laser) and 505-550 nm emission band.

(ix) Antiviral activity in EIAV-infected ED fibroblasts.

Both equine dermal fibroblasts (ED) and Equine Infectious Anemia Virus(EIAV), Wyoming isolate (Malmquist et al., 1973) were gifts from Profs.A. Yaniv and A. Gazit, from the Sackler Medical School of the Tel-AvivUniversity, Israel. ED cells were plated on plastic 6-well tissueculture plates (Nunclone) at density of 5×15 cells per well in 2 ml ofDMEM/10% FCS (fetal calf serum). The medium was removed after 12-20hours and the cells were inoculated for 2 hours with 0.5 ml DMEM/10% FCScontaining 5×10⁶ pfu (plaque forming units) of EIAV and 10 μg/ml ofpolybrene (hexadimetrine bromide, Sigma) (Carpenter and Chesebro, 1989).Following the incubation, another 2.5 ml of DMEM/10% FCS was added toeach well. On the following day, the medium was discarded and wassubstituted for 2 ml of DMEM/10% FCS, containing 0.5 μCi/ml of 5,6−³Huridine (Amersham). Different concentrations of the conjugates wereadded to the wells in duplicates. Every 34 days, 1.2 ml samples of themedium were collected and replaced with same amount of medium,containing radioactive uridine and the tested. The samples wereclarified from cell debris by spinning at 12000 rpm for 5 minutes on theEppendorf centrifuge. Viral particles from the clarified supernatantswere collected by ultracentrifugation at 75000 rpm, 4° C. on the BeckmanTL 100 centrifuge using TLA 100.2 rotor (Cheevers et al., 1977). Thesupernatants were carefully discarded and the pellet was resuspended in200 μl of 1% SDS/5% Tritone X-100, mixed with 1 ml Ultima Goldscintillation liquid (Packard) and counted on LS 1701 Brukerscintillation counter. Typical duration of the experiment was 15 days;EIAV growth curves in the presence of various concentrations of theconjugates in the medium were obtained. Development of the cytopathiceffect (cpe) in the infected ED cells was monitored by light microscopy.

(x) Anti-HIV assay.

The HIV-1 strains NL4-3 and Ba-L and the CD4⁺lymphocytic cell linesSUP-T1, MT-4 and MT-2 and P4-CCR5 MAGI cells were obtained from the MRCAIDS reagent program or the NIH, AIDS Research and Reference ReagentProgram.

T-tropic HIV-1 laboratory strains IIIb, 2D as well as AZT-resistant andUC781-resistant strains were utilized in this study, along with clinicalisolate (clade C). HIV-1 clinical isolate, resistant to AZT is agenerous gift from Dr. Mark A. Wainberg, McGill AIDS Center, McGillUniversity, Montreal, Canada. HIV-1 laboratory strain (IIIB) resistantto UC781 was developed in vitro by increasing concentrations of UC781 to100×IC₅₀ as described previously (Borkow et al., 1999).

MT-2 or MT-4 cells were cultured in RPMI 1640 supplemented with 10%fetal calf serum (FCS), 0.2 mM glutamine andpenicilline/streptomycin/nistatin mixture. The cells were propagated in96-well tissue culture plates at the density of 5×10⁴ cells per well.Peripheral mononuclear blood cells (PMBC) were separated from wholeblood samples by centrifugation in Ficoll gradient for 10 min at 400 g.PMBC were cultured in the same medium in 24-well tissue culture platesat the density of about 10⁶ cells per well.

Anti-HIV activity and cytotoxicity measurements in MTA cells were basedon viability of cells that had been infected or not infected with HIV-1exposed to various concentrations of the test compound. The cells wereinfected by incubation with different titers of the HIV-1 strains for 2hrs at 37° C. in the presence or absence of the AAC of the invention.Then the cells were washed from the virus by centrifugation at 400 g andthe supernatants were discarded. The cells were resuspended in culturemedium, supplemented with different concentrations of the testcompounds. The cells were allowed to proliferate for 5 days, the numberof viable cells was quantified by a tetrazolium-based colorimetricmethod (CellTiter 96® AQ_(ueous) One solution Cell Proliferation Assay,Promega). Anti-HIV activity in P4-CCR5 MAGI cells was done as follows:cells (1×10⁵/ml) were infected with 10 ng/ml of p24 antigen of HIV-1Ba-L in the presence of varying concentrations of the test compound. 24hours post infection, cells were washed twice with PBS and resuspendedin the medium containing the appropriate drug concentration. Five daysafter infection the cells were washed with PBS and evaluated forβ-galactosidase activity.

(xi) Flow cytometry analysis

Measurement of chemokine receptors CXCR4, CCR5 and the CD4 receptor onphytohemagglutinin (PHA)-activated PBMC was performed by flow cytometryanalysis. Briefly, 0.5×10⁶ cells were washed in ice-coldphosphate-buffered saline (PBS) and incubated for 30 minutes at 4÷C.with fluorescent labelled mAb 12G5 and 2D7 and Leu3a (Becton Dickinson,San Jose, Calif.) or with isotype control mAbs in the presence orabsence of the test compound. Then, the cells were washed with ice-coldPBS and were fixed in PBS containing 1% formaldehyde. For each sample,10,000 events were analysed in a FACScalibur™ system (Becton Dickinson).Data were acquired and analysed with CellQuest™ software (BectonDickinson).

(xii) Measurement of intracellular calcium concentration

The intracellular calcium concentrations [Ca²⁺]_(i) were determinedusing the following procedure: SUP-T1 cells or THP-1 were loaded withFluo-3 (Sigma, St. Louis, Mo.). Fluorescence was measured in aFluoroskan Ascent fluorometer (Labsystems, Helsinki, Finland). Cellswere first stimulated with dilution buffer (control) or test compound atdifferent concentrations. As a second stimulus, SDF-1α (20 ng/ml) orRANTES (1 μg/ml) were used to induce [Ca²⁺]_(i) increase. The secondstimulus was added 10 sec after the first stimulus. The compoundconcentration required to inhibit the [Ca²⁺]_(i) increase by 50%(IC_(50[Ca2+]i)) was calculated.

(xiii) Virus-binding assay.

MT4 cells (5×10⁵) were incubated with supernatant containing 1×10⁵ pg ofp24 antigen of wild type HIV-1 in the presence of differentconcentrations of the test compound. One hour after infection, cellswere washed 3 times with PBS and p24 antigen bound to the cells wasdetermined by a commercial ELISA test (Coulter, Spain).

(xiv) Reporter gene transactivation inhibition

Transactivation inhibition is measured in HeLa cells transfected withtwo plasmids, HIV LTR-reporter gene construction and Tat proteinexpression vector. Reporter gene expression is dependent on Tat bindingto TAR element while transcription from LTR promotor occurs. The levelof transactivation is determined by measuring the activity of thereporter gene product by its characteristic reaction, for example,chloramphenicol acetyl transferase (CAT) acetylates chloramphenicol(Kessler and Mathews, 1992). Cell extracts containing CAT are incubatedwith ¹⁴C-labeled chloramphenicol in the presence of acetyl coenzyme A,extracted with ethyl acetate and resolved by thin layer chromatography(TLC). Results are observed as spots of acetylated and non-acetylatedantibiotic after autoradiography of TLC plates (e.g. Calnan et al., 1991a, b). Incubation of the cells after transfection in the presence ofvarious concentrations of the aminoglycoside conjugate in cell culturemedium and with further treatment as described above, a decrease ordisappearance of acetylated chloramphenicol spots on TLC suggestinhibition of transactivation by the added conjugates.

xv) Inhibition of Tat-induced Kaposi sarcoma and endotelial cellsproliferation.

Kaposi sarcoma (KS) cells, obtained from biopsy of KS lesions of AIDSpatients, and human umbilical vascular endothelial cells (HUVEC) werecultured in RPMI 1640 medium supplemented with 10% FCS and conditionedmedium, obtained from HTLV-II infected T-cell lymphoma culture medium asdescribed (Salahuddin et al., 1988; Fiorelli et al., 1999). Cells areseeded at 10⁴ cells/well in 96-well plate, coated with gelatin. After 24hrs Tat protein (10 ng/ml-1 ng/ml), 0.5-1 μCi [³H] thymidine (Amersham)and different concentrations of an AAC of the invention are added perwell. Cells are harvested after 72 hrs and radioactive thymidineincorporation is measured using a beta counter.

(xvi) Crystallization trials.

Synthetic 31-nucleotide TAR RNA oligonucleotide (5′-GGC CAG AUC UGA GCCUGG GAG CUC UCU GGC C-3′) containing sequence 18-44 of HIV-1 LTR,purified by PAGE and reverse-phase HPLC (Dharmacon, Inc) is used forcrystallization trials. Crystallization efforts of TAR RNA will startwith the use of Hampton Research crystallization screens beginning withthe 48 conditions of the Natrix™ (nucleic acid sparse matrix). Theseinvolve varying of pH in the range of 5.6 to 8.5, using variouspolyethylene glycols, isopropanol, MPD and 1,6 hexanediol asprecipitants and various mono and divalent cations as additives. Anadditional Hampton Research screen (nucleic acid mini screen) with MPDas precipitant, pH range of 5.5 to 7.0, polyamines as cobalt hexamineand spermine and chlorides of mono- and di-valent cations as additives,will be employed as well. Once we obtain crystals, their quality aspotential X-ray diffracting candidates will be improved through minutevariation of the crystallization conditions like temperature,crystallization method (sitting drops, hanging drops), speed of crystalformation and others. Nucleic acids have a built-in advantage overproteins in their ability to incorporate Br or I atoms in the 5 positionof uracil. X-ray diffraction of RNA containing 5-Br uracil or 5-I uracilcan provide phasing information used by the SIR (single isomorphousreplacement), SIRA (single anomalous replacement) or MAD (multipleanomalous diffraction) for structure solution. Once successfulcrystallization conditions are established, the Br and I uracilderivatives of TAR RNA will be prepared, crystallized and subjected toX-ray diffraction collection in order to provide the phasing needed forobtaining a good electron density map.

EXAMPLE 11.

Monosaccharide conjugates with acetamidino and guanidino compoundsinhibit Tat binding to TAR

In order to determine which one of the side chains attached to amonosaccharide would display a better affinity to TAR RNA, severalderivatives of methyl α-D-mannopyranoside (MMP), compounds 4 and 7-12,were tested for their affinity to TAR on an L-arginine-Amberlite column,and their potential inhibition of Tat binding to TAR, using thegel-shift assay as described above. The results are presented in theTable 1. The gel-shift assays (FIG. 3) demonstrated that compounds 10and 11 inhibited the binding of 60 nM Tat R52 peptide to 6 nM TAR RNAwith CI₅₀ values of 9 and 11 mM, respectively (Table 2). These CI₅₀values were normalized to the conditions presented by Frankel et al.(Tao and Frankel, 1992), giving apparent K_(i) values of around 1 mM,suggesting that compounds 10 and 11 are stronger inhibitors of Tat-TARbinding than the free arginine, and are similar to L-argininamide andagmatine (Tao and Frankel, 1992), and to neomycin (Zapp et al., 1993).All other monosaccharide derivatives did not show significant inhibitionof Tat binding to TAR (Table 2). These results indicate that a acompound comprised of a carbohydrate “core” attached to a singlestrongly basic group with the geometry resembling that of guanidine, mayserve as a specific inhibitor of Tat binding to TAR.

The gel-shift assay revealed that compound 12 inhibited the Tat-TARinteraction with a CI₅₀ of 1.8 mM which corresponds to an apparent K_(i)of approximately 160 μM (normalized as above) (Table 2). CI₅₀ (or K_(i))values (FIG. 3A and Table 2) obtained for compounds 10, 11 and 12,suggest, that not only a carbohydrate core facilitates the binding toTAR RNA of inhibitors, containing guanidinium or acetamidinium groups,but a chain, such as that of arginine, connecting the core and thecharge-bearing moiety, is beneficial.

TABLE 1 Concentrations of the compounds that elute 50% of TAR RNA (CE₅₀)from L-arginine-Amberlite affinity column. Compound CE₅₀, mM Tat R52peptide 0.5 Arginine HCl 1500 6-cyano MMP (4) 1200 6-amino MMP (7) >15006-amido MMP (8) >1500 6-amidoximo MMP (9) >1500 6-guanidino MMP (10) 6506-(N-acetamidino) MMP (11) 730 RMMP (12) 310 R4K (13) 2.5 R3G (14) 1.4

EXAMPLE 12.

Binding of aminoglycoside-arginine conjugates to TAR RNA

The ability of aminoglycoside-arginine conjugates to bind TAR RNA usingL-arginine-Amberlite affinity column is shown in Table 1. This resultsuggests that the aminoglycoside-arginine conjugates bind to TAR RNAwith similar efficiency as the Tat peptide and compete for the samearginine-binding site on TAR.

The molecular weights of the aminoglycoside-arginine conjugates (ca 940,1076 and 1109 Da for R3G (14), R4GC_(1a) (15) and R4K (13),respectively) are close to that of the Tat R52 peptide (1434 Da), thustheir binding to TAR RNA was clearly observed as electrophoretic bandshifts (FIG. 3). All the conjugates displayed high affinity to TAR RNA.

TABLE 2 Concentrations of the compounds that displayed 50% inhibition(CI₅₀) and 50% binding (CD₅₀) in gel-shift experiments on TAR RNA.Compound CI₅₀, mM CD₅₀, nM TAR RNA, nM Tat R52 peptide 6-12 2 Tat R52peptide 35 6 Tat R52 peptide 75 12 Tat R52 peptide 200 20 Arginine HCl 42 Arginine HCl 38 6 6-cyano MMP (4) 46 6 6-amino MMP (7) 56 6 6-amidoMMP (8) >50 6 6-amidoximo MMP (9) >50 6 6-guanidino MMP (10) 9 66-(N-acetamidino) MMP (11) 11 6 RMMP (12) 1.8 6 R4K (13) 2500 12 R3G(14) 500 12 R4GC_(1a) (15) 200 20

The CD₅₀ of R4K (13) to 12 nM ³²P-labeled TAR (in the gel-shift assay)is 2.5 μM (FIG. 3B, Table 2). The band shifts observed for the R4K (13)complex with TAR remained unchanged at increasing concentrations of R4Kin the range of 1 to 5 μM. Since a Tat-TAR complex has a 1:1stoichiometry and the band shift of R4K-TAR complex was observed to beapproximately twice than that of Tat R52-TAR one (FIG. 3B), a 2:1 ratiois suggested for the R4K conjugate to RNA in the complex.

The R3G (14) conjugate displayed binding to 12 nM of TAR with a CD₅₀ ofca 500 nM (FIG. 3C, Table 2). The gel mobility of R3G-TAR complexesdeclined significantly with the increase of R3G concentration in therange of 0.5 to 4 μM, as was observed from the increase of the bandshifts and an increasing precipitation of the complexes in the wells(FIG. 3C). At the same concentration of TAR (12 μM), the CD₅₀ of Tat R52was found to be 75 nM (Table 2).

The R4GC_(1a) (15) conjugate formed a complex with 20 nM of TAR RNA atconcentrations starting from 200 nM. The CD₅₀ of Tat R52 at the sameconcentration of TAR RNA was 200 nM. By comparing the gel shift ofR4GC_(1a) (15) -TAR to that of Tat R52, it is suggested that the ratioof R4GC_(1a) to the RNA in the complex is 1:1 (FIG. 3D, Table 2). Anapproximate linear dependence of Tat R52 CD₅₀ values on TAR RNAconcentrations was observed (Table 2). We have normalized the CD₅₀values obtained in our experiments to the reported conditions (Calnan etal., 1991 b), and apparent K_(d) values, suitable for comparison withreported data are available. The apparent K_(d) values for the conjugatecomplexes with TAR RNA were found to be 416 nM for R4K, 83 nM for R3G,and approximately 20 nM for R4GC_(1a). Thus, a trisaccharide corebearing three or four arginine residues can successfully mimic thebinding pattern of Tat.

EXAMPLE 13.

Determination of the conjugates' binding sites on TAR RNA

a. RNase A footprinting

TAR RNA and RNase A concentrations were established by co-titration. Toproduce a “single-hit” kinetics conditions, 1 μM TAR versus 200 pM RNaseA were selected. Tat R52 and AAC concentrations were determined bytitration. Effective protection of 1 μM TAR was observed with 2 μM TatR52, 10 μM of R4K and 4 μM R3G. GB4K did not exhibit binding to TAR RNA,neither in gel-shift experiments (up to 50 μM) nor in the protectionexperiments (at 40 μM). RNase A cleaved TAR RNA in an uneven manner(FIG. 4A., lane 2). The most susceptible regions for cleavage were thesingle-stranded loop, nucleotides U31-G34 as well as C24 and U25 of thebulge. Strong cleavage was also observed at G21-G41 and A20-U42 pairs(lower stem). Binding of R52 (2-4 μM) caused a weakening of the bands inthe upper stem region, (G26-C39, A27-C38, as well as U40) (FIG. 4A.,lanes 3, 4). At 4 μM R52, the cleavage at G34 (loop) was enhanced, aswell as a certain enhancement of bands A20, G21, U23 and U25 wasobserved. Remarkably, the band corresponding to the R52-TAR complexstill was observed on the gels even after electrophoresis under stronglydenaturing conditions. In the presence of R4K (10-20 μM), the upper stem(G26-C29), the lower stem and the bulge (G21-U23, partially C24 and U25)were protected as well as C38, C39 and U40. Cleavage at C19 and A20 ofthe lower stem and C30, G33 and G34 of the loop was enhanced by 10 μMR4K, whereas with 20 μM R4K the protection of C30, G34 and A20-G21 wasobserved (FIG. 4A., lanes 5, 6). R3G (4-8 μM) displayed similarbehavior; protection of the lower stem and the bulge was more pronouncedwith 8 μM R3G than with 20 μM R4K. Cleavages at C19-A20 in the lowerstem as well as at G30 and G34 in the loop were observed (FIG. 4A.,lanes 7, 8). In the presence of GB4K (20 μM) no significant protectionwas observed. With 40 μM GB4K, U25 (bulge), U31, G33, A35 (loop) as wellas nucleotides 37-42 were partially protected, whereas the bands of A20,C30 and G34 were partially enhanced (FIG. 4A., lanes 9, 10).

b. Uranyl nitrate photoprobing of TAR RNA

UO₂ ⁺⁺has high affinity to DNA and RNA backbone phosphates. Irradiationof the uranyl-nucleic acid complex with light, at a wavelength of 420 nmleads to the oxidation of a proximal deoxyribose/ribose ring, resultingin a cleavage of the backbone (Nielsen et al., 1992). Slightly acidicconditions (pH 6.5 and lower) modulate the uranyl cleavage, reflectingthe conformational changes of nucleic acids. For probing the conjugates'interactions with TAR RNA, the best results were obtained in cacodylatebuffer pH 6.5, 25-50 μM uranyl nitrate and 10-20 min irradiation. 1 μMTAR was cleaved by 50 μM uranyl nitrate after 10 min irradiation, in anuneven manner. Nucleotides of the lower stem (C18-G21 and U40) and ofthe loop (U31 and G33) were better cleaved by the uranyl cation thanthose of the upper stem (G26-C29). The bulge (U25 and C24) washypersensitive to cleavage (FIG. 4B., lane 2).

In the presence of Tat R52 (4 μM), protection of the loop and the upperstem (A27-A35) as well as U38-G44 (the opposite strand) was observed,but no changes were observed in the bulge region. Cleavage at C18 andG21 was enhanced (FIG. 4B., lanes 3, 4). With 10-20 μM R4K (FIG. 4B.,lanes 5, 6) and 4-8 μM R3G (FIG. 4B., lanes 7, 8) the whole TAR RNAmolecule appeared protected against cleavage. GB4K (20 μM), under thesame conditions, did not affect TAR RNA uranyl photocleavage (FIG. 4B.,lanes 9). 40 μM GB4K displayed protection of the upper stem (A27-C29),C39-G44 of the opposite strand and the loop (C30-A35).

c. Lead acetate footprinting

Pb⁺⁺cations cause cleavage predominantly of phosphoester bonds of RNAsingle-stranded regions. Cleavage also may occur in double-strandedregions if they contain weak, bulged or destabilized base pairs (Walliset al., 1997). Lead acetate footprinting was found to be a suitable andaccurate method for the studies of AAC binding to TAR. In the presenceof R52, A22-U40 and G26C39 pairs were protected as well as C19, A20, A27and G28 (FIG. 4C, lanes 3, 4). Protection was also observed at G33 andG34 of the loop. In the presence of AAC, the protection of the bulgeregion with flanking base pairs was observed, along with some protectionat G34 of the loop (FIG. 4C, lanes 5-8).

EXAMPLE 14.

Specificity of binding of aminoglycoside-arginine conjugates to TAR RNA.

Binding of R3G and R4K to a variety of short RNA oligonucleotides,including rG₁₅, rG₁₅-rC₁₅ duplex and truncated TAR RNA sequences, wastested by gel-shifts. The conjugates did not display any binding tothese RNAs in the micromolar concentration range (data not shown).Binding competition experiments were performed with ³²P labeled TAR RNAin the presence of various amounts of unlabeled yeast tRNA (Sigma). Ingel-shift experiments, a ten-fold excess of tRNA did not inhibit complexformation between TAR and R52 or the conjugates (FIGS. 7A, 7B) Ahundred-fold excess of tRNA inhibited complex formations (over 90%inhibition, data not shown). This demonstrates similar specificity ofthe binding of Tat R52 and AAC to TAR RNA. In the “single-hit” RNase Afootprinting, the presence of a ten-fold excess of yeast tRNA in thereaction mixtures did not affect the protection pattern significantly.However, a trend of AAC binding inhibition to definite positions on TARunder these conditions was quite obvious (FIG. 7C). Densitometryquantitations, as described above, showed these positions to be G21-U25,G28 and U38 (FIGS. 7D, 7E). Positions not affected by tRNA excessresemble Tat R52 binding site (FIG. 6, FIG. 7E).

EXAMPLE 15.

Binding sites of AACs of the invention on TAR RNA.

Gels were analyzed by densitometry using a “Storm 820” phosphoimager,and the intensities of the bands were determined by the ImageQuantprogram. The results of the measurements are presented in FIG. 5 as therelative intensity of each band to the respective control band. Zeroline stands for the abscence of any effect (within 10% experimentalerror), positive peaks denote protection of the nucleotide from cleavageand negative peaks represent cleavage enhancement (by RNase A) atcertain positions due to conformational changes of TAR RNA. From thesegraphs, the binding sites for R52 and AAC were derived (FIG. 6).

Based on the footprinting results (FIGS. 4-6), we can suggest that R52makes contacts with TAR at the G26-C39 pair, the base-pair that wasdetermined to be crucial for TAR major groove recognition by ligands(Gelus et al, 1999); contacts with A27, G28 and G40 were also assigned.The whole region between the bulge and the loop acquires a certainburied conformation (as can be concluded from uranyl footprintingresults). Binding of R52 causes conformational changes in the bulge,making U23 and U25, as well as A20 and G21 more accessible to RNase Acleavage. In the loop, G34 and A35 become more exposed to RNasedigestion, whereas in uranyl and lead footprinting experiments, G34 isfound to be protected. G34-G33, shown later on, is a metal-binding site.The results of our footprinting experiments suggest that the bindingsite for R52 (FIG. 6) is similar to that for Tat peptide and inaccordance to a model of Tat peptide-TAR RNA complex (Seewald et al.,1998).

The binding of an AAC of the invention to TAR differs significantly fromR52 binding, while R4K and R3G bind similarly to TAR (FIGS. 4-6). Thecontacts with TAR can be assigned at the A22-U40, G26-C39 and A27-U38pairs (A27 with R4K) as well as at G21. The whole bulge region in eachcase is involved in the binding. C19 and A20 (with R3G) are protected inUO₂ ⁺⁺and Pb⁺⁺footprinting experiments, while they are readily cleavedby RNase A. C30, U31(only with R3G), G33 (only with R4K) and G34 aremore exposed to RNase A digestion upon AAC binding, whereas in uranyland lead footprinting, there is protection at G34 with both compounds.This behavior resembles R52 binding to TAR. Molecular weights of AAC aresmaller then of R52, and, assuming R52 is folded as β-hairpin, similarlyto the described model of Tat basic nonapeptide bound to TAR (Seewald etal., 1998), we can suggest that an AAC occupy approximately ⅔ of thepeptide molecular volume. AAC makes more contacts with TAR than R52(FIG. 6); thus it is plausible that there are two molecules of AAC inthe complex with TAR RNA. This suggestion is supported by the gel-shiftsand the fact that the whole TAR RNA molecule in the presence of AAC isprotected against UO₂ ⁺⁺cleavage (uranyl footprinting under ourconditions reflects conformational changes in RNA). In contrast, uponR52 binding, only the upper stem part of TAR is protected. There is acertain similarity in AAC protection of TAR RNA to that of neomycin B,which protects the C19-G43 and A22-U40 pairs of the lower stem, U23 andU25 of the bulge and G26 of the upper stem (Wang et al., 1998). Thus, wecan conclude that an AAC of the invention possesses bothaminoglycoside-like and peptide-like features of binding to TAR. Wesuggest that one of the AAC molecules binds in the lower stem-bulgeregion, whereas the second one binds in the upper stem, similarly toR52, but inducing different conformational changes in the TAR RNA loop(FIG. 6).

It was unexpected that GB4K, which is very similar to R4K, differs sodrastically in TAR RNA binding. GB4K is a very weak TAR RNA binder andpresumably forms only non-specific electrostatic contacts with TAR.Nevertheless, in the presence of GB4K, U38-U42 are found to be protectedas well as U25, U31 and G33. Conformational changes upon GB4K binding inTAR, similarly to AAC, induce cleavages by RNase A at A20, C30 and G34,which is probably a general feature of binding of this class of ligandsto TAR RNA (FIG. 6). We conclude that in the context ofaminoglycoside-based peptidomimetic TAR binders, not any guanidinebearing side-chains are suitable for the efficient recognition; so faronly arginine residues were properly recognized by TAR RNA. It is worthnoting that streptomycin, bearing two guanidino groups on thedeoxystreptamine ring, binds to TAR (Wang et al., 1998), but is not anefficient inhibitor of the Tat-TAR interaction (Mei et al., 1995). Wesuggest that the α-amino groups of the conjugated arginine moieties areessential for this interaction.

Previously, we suggested that TAR forms the complex with two moleculesof R4K (FIGS. 3B, 7A,). The difference in band shifts in the presence oftRNA (FIG. 7B) may indicate that the TAR-R4K complex, under thiscondition, contains only one molecule of R4K. The same may be true forthe R3G-TAR complex, whose band shifts are more complicated forinterpretation, since R3G is a mixture of isomers. These observationssupport our suggestion that R4K and R3G bind to at least two sites onTAR RNA, which are different in affinity. These sites could bedistinguished by RNase A footprinting in the presence of an excess oftRNA. The AAC protection pattern in the presence of tRNA tends to besimilar to that of R52, while the protection of G21-U25 was inhibited(FIG. 7D). The site with higher affinity involves the bulge and theflanking base pairs, similar to the Tat peptide (like R52) binding site,while the low affinity site is in the lower stem-bulge region, similarto neomycin B binding site (FIG. 7E, FIG. 12). It is worth noting thatID NMR spectra taken at different ratios of R4K to TAR suggest that R4Kpreferentially binds first to one, then to the second site on TAR (incollaboration with Professor T. L. James, UCSF, unpublished results).

EXAMPLE 16.

Cytotoxicity measurements

All the compounds were tested for their toxicity in the HeLa, LAN-1 andED cell lines. None of the compounds up to concentrations of 1 mM causedany cell growth inhibition, as measured by ³H-thymidine incorporation.

EXAMPLE 17.

Intracellular accumulation and distribution of fluorescent-labelledconjugates.

An R4K fluorescent derivative (R4K-fluorescein) was prepared and itsuptake by rat neurons (FIG. 9) and human peripheral blood mononuclearcells (FIG. 10) was demonstrated. Similar experiments were performedusing equine dermal fibroblasts (ED cells), both uninfected and infectedwith EIAV. After 30-60 min incubation with 10 μg/ml of either R4K-FITCor R3G-FITC, an intense fluorescence was observed in the cell nuclei(FIGS. 11B, 11D). This indicates that the conjugates efficientlypenetrate into the ED cell nuclei of uninfected as well as EIAV-infectedcells.

EXAMPLE 18.

Antiviral activity of R3G and R4K conjugates.

18 a. EIAV-infected ED cells.

Equine infectious anemia virus (EIAV) of Wyoming strain is adapted togrow in equine dermal fibroblasts (ED), without damaging them with atiter of 1 μfu per cell (Malmquist virus). We used a significantlyhigher infection level (superinfection), around 10 pfu per cell, inorder to accelerate the viral growth and to cause the development of theEIAV-induced cytopathic effect (cpe) in the cells at the late stage ofthe infection. In the absence of inhibitors, EIAV proliferated in the EDcells reaching a plato 9-12 days after infection (FIG. 8). After 15 to17 days, the viral titer in the medium dropped and the cells appeared ascpe phenotype (FIG. 8C): cell nuclei were condensed, vacuoles wereobserved in the cytoplasm and the cells formed syncytia. Development ofcpe started 10-12 days after infection, reaching a maximum on days15-17, followed by rapid cell death and exfoliation from the plasticbottom of the well.

Addition of 50-100 μM of R4K and 12.5-50 μM of R3G to the EIAV-infectedcells caused a significant (3-5 fold) inhibition of the viral growth ina dose-dependent manner (FIG. 8). After day 12, a slight increase of theEIAV titer was observed. GB4K (100-250 μM) did not show any effect onthe viral growth. Toxicity of the conjugates for ED cells was tested by³H-thymidine incorporation as described above. The compounds, up to 1μM, concentration did not inhibit DNA synthesis in the cells.

A noticeable inhibition of the cpe development was observed at 25 and 50μM R3G, even after 13-15 days (FIGS. 8C, 8D). Untreated cells at thesame time displayed cpe development, whereas cell treated by R3Gpreserved normal phenotype. This observation correlates with the viralproduction. Moreover, when R3G was added to the infected cells duringthe cpe onset (day 12-13), at day 15 the development of cpe wasinhibited and cell damage was not noticeable. The viral titer decreasedat least twice compared to control. At the same time, untreated cellsdeveloped massive cpe phenotype and were significantly damaged by day 15(data not shown).

18 b. HIV-infected human cells.

The activity of AACs of the invention against HIV-1 NL4-3, RF, and NL4-3AMD3100-resistant T-tropic strains, T-tropic HIV-1 AZT-resistantclinical isolate AOM as well as M-tropic HIV-1 Ba-L strain are presentedin Table 3.

The 50 % cytotoxic concentrations (CC₅₀) were found to be >1130 μM forR4K and >3940 μM for R3G. R3G and R4K inhibited HIV-1 NL3-4proliferation at 50% effective concentrations (EC₅₀) of 15 μM and 31 μM,respectively. The chemokine stromal-derived factor SDF-1α was activeagainst HIV-1 NL4-3 at EC₅₀ of 0.04 μM.

R3G was active against T-tropic HIV-1 RF strain and AOM clinical isolateas well as M-tropic HIV-1 Ba-L strain at EC₅₀ of 16-35 μM.

The NL4-3 AMD3100-resistant virus was cross-resistant to SDF-1α(>25-fold) to R4K (>4-fold) and R3G (>9-fold), suggesting that thesecompounds share a similar mode of action.

TABLE 3 Anti-HIV-1 activity of the AAC, AZT and the CXC-chemokineSDF-1α. EC₅₀ ^(a) (μM) HIV-1 HIV-1 HIV-1 HIV-1 AMD HIV-1 CC₅₀ ^(b)Compound NL4-3 RF AOM 3100res Ba-L^(c) (μM) R4K 31 >113 67 >113 >1131130 R3G 15 35 16 >133 29 >3940 SDF-1α 0.04 — — >1 — >0.1 AZT 0.01 0.023.7 0.005 0.2 >7.5 ^(a)EC₅₀: 50% effective concentration, orconcentration of the compound required to inhibit HIV-1 replication by50%, as measured by the MTT assay. ^(b)CC₅₀: 50% cytotoxicconcentration, or concentration of the compound required to reduce theviability of MT-4 cells, as measured by the MTT assay. ^(c)EC₅₀: 50%effective concentration, or concentration of the compound required toinhibit HIV-1 replication by 50%, as measured by β-galactosidaseactivity in P4-CCR5 cells infected with the HIV-1 Ba-L strain.

The toxicity of the conjugates R3G and NeoR to MT-2 cells as well asPMBC lines ETH, derived from Ethiopian blood sample and IS, derived fromIsraeli blood sample (healthy controls) was determined after 2 daysincubation of the cells in the presence of the conjugates. 50% cytotoxicconcentrations (CC₅₀) of the conjugates were determined to be >500 μMfor R3G and 250 μM for NeoR (Table 4). The activity of the AAC againstHIV-1 IIIb and 2D laboratory strains as well as against AZT resistant,UC781 resistant HIV-1 strains and clade C HIV-1 clinical isolate, arepresented in Table 4. The 50% effective concentrations (EC₅₀) of R3G andNeoR were 6-10 μM and 3-4 μM, respectively.

TABLE 4 Anti-HIV-1 activity of AAC. HIV-1 HIV-1 HIV-1 HIV-1 HIV-1 IIIB2D AZTres^(a) UC781res^(b) Clade C^(c) Compound EC₅₀ (μM)^(d) CC₅₀(μM)^(e) TI₅₀ ^(f) R3G 10 10 8 6 6 500 50-85 NeoR 3 4 3 3 3 250 60-85^(a)HIV-1 clinical isolate, resistant to AZT. ^(b)HIV-1 laboratorystrain (IIIB) resistant to UC781. ^(c)HIV-1 clinical isolate (clade C).^(d)The 50% effective concentration. Inhibition of HIV-1 replication wasdetermined by assessing syncytium formation and by a tetrazolium basedcolorimetric assay (CellTiter 96 ® Aq_(ueous) One solution CellProliferation Assay, Promega). The data is the average of twoindependent experiments done in duplicates. ^(e)The 50% cytotoxicconcentration was determined by a tetrazolium based colorimetric assayas in ^(d) above. The data is the average of two independent experimentsdone in duplicates. ^(f)The 50% in vitro therapeutic index (ratioCC₅₀/EC₅₀).

MT-2 cells were infected with different dilutions of HIV-1 clade C or 2Dstrains (1: 1 to 1:16) and incubated for 4 days with 10-20 μM R3G or5-10 μM NeoR (FIG. 13). Cells infected with HIV-1 clade C developed morethan 50% cpe, whereas cells infected with 2D strain developed onlyaround 25% cpe at the same period of time. The efficacy of the AAC ofthe invention is significantly increased with viral dilutions. Atearlier stage of infection (2D strain, 25% cpe) the inhibition by theAAC was significantly more pronounced than at later stage of theinfection (clade C, >50% cpe).

To determine the effect of the AAC of the invention on viralinfectivity, the infection of MT-2 cells by HIV-1 was performed in thepresence and absence of 20 μM R3G or 10 μM NeoR (FIG. 14). When thecells were infected with HIV-1 clade C in the presence of the AAC andthen incubated for 3 days in the absence of the AAC (reaching 25% cpe),the virus growth was inhibited by 30% for R3G and 60% for NeoR (FIG.14). Presence of the AAC during infection depressed viral proliferation.When the cells were incubated with the AAC after the infection, theantiviral effect of the conjugates was significantly increased (FIG.14).

The effect of the AAC in the presence of AZT was tested in HIV-1 2D andclade C. The antiviral effect of both R3G and NeoR seems to be additiveto that of AZT (FIG. 15).

Interaction with chemokine receptors.

In order to elucidate whether the anti-HIV activity ofaminoglycoside-arginine conjugates is due to their interaction withCXCR4, we have tested their capacity to inhibit the binding of a mAb toCXCR4 (12G5). SDF-1α, the natural ligand of CXCR4, was used forcomparison. Table 5 shows the concentrations of 50% inhibition (IC₅₀) of12G5 mAb binding (IC_(50-12G5)) by R3G, R4K, NeoR and SDF-1α. Theconjugates R3G, R4K and NeoR showed high affinity for CXCR4 (as measuredby the inhibition of 12G5 binding to SUP-T1 cells or PMBC) which isconsistent with their anti-HIV activity. None of the compounds inhibitedthe binding of 2D7, a monoclonal antibody directed to CCR5 or ananti-CD4 antibody (Leu3a) in IL-2/PHA-stimulated PBMC.

TABLE 5 Inhibition of anti-CXCR4 mAb (12G5) binding to CXCR4⁺ cellsIC_(50-12G5) ^(a) (μM) Compound SUP-T1 cells PMBC R4K 3.7 2.2 R3G 7.72.7 NeoR — 2.4 SDF-1α 0.013 — ^(a)IC_(50-12G5): 50% inhibitoryconcentration, or concentration of the compound required to inhibit by50% the binding of 12G5 mAb to CXCR4⁺ cells. —: not tested.

We have found that in the presence of 5 μM NeoR, the binding ofanti-CXCR4 monoclonal antibody (mAb) 12G5 to PHA-activated PMBC wassuppressed significantly more than in the presence of 5 μM R3G (FIG.16A). The effect of NeoR (5 μM) on binding of 12G5 mAb to CXCR4 ispresented in FIG. 16B. The effect of R3G (25 μg/ml) and R4K (25 μg/ml)on the binding of 2D7 mAb to CCR5, 12G5 mAb to CXCR4 and Leu3a mAb toCD4 in PHA-stimulated PBMC is presented in FIG. 17.

To further evaluate the interaction of aminoglycoside-arginineconjugates with CXCR4, we tested the capacity of SDF-1α to induce anintracellular Ca²⁺signal in the presence of these conjugates. Both R3Gand R4K inhibited the SDF-1α-dependent Ca²⁺signal in a dose dependentmanner (FIG. 18). R3G and R4K did not inhibit the intracellularCa²⁺signal induced by RANTES in THP-1, CCR5⁺cells (data not shown).

Inhibition of virus binding to CD4⁺cells.

Our experiments showed that R4K and R3G inhibit the binding of HIV-1NL4-3 to MT-4 cells in a dose dependent manner. Dextran sulfate was alsoactive, while SDF-1α at the concentration of 0.5 μg/ml did not inhibitthe binding of HIV-1 to MT-4 cells (FIG. 19). Similarly, R4K and R3Ginhibited the binding of the R5 strain Ba-L to MT-4 cells in a dosedependent manner (data not shown)

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What is claimed is:
 1. A conjugate of the formula:

wherein A is CH₃ or NH₂; X is a linear or branched C₁-C₈ alkyl chainoptionally containing hydroxy, amino and/or oxo groups; n is an integerfrom 1 to 6, and Sac is the residue of a mono- or oligo-saccharide,provided that when A is NH₂ and X is —(CH₂)₃—CH(NH₂)—C(=O)—, themonosaccharide residue is not substituted at the position 1, and n is aninteger from 2 to 6 when Sac is the residue of an oligosaccharide.
 2. Aconjugate according to claim 1, wherein A is CH₃.
 3. A conjugateaccording to claim 2, wherein Sac is the residue of a monosaccharide. 4.A conjugate according to claim 2, wherein Sac is the residue of anoligosaccharide.
 5. A pharmaceutical composition comprising atherapeutically effective amount of a compound according to claim 1, anda pharmaceutically acceptable carrier.
 6. An antiviral pharmaceuticalcomposition according to claim 5 in unit dosage form wherein saidtherapeutically effective amount is an antiviral-effective amount.
 7. Anantiretroviral pharmaceutical composition according to claim 6, whereinsaid antiviral effective amount is an antiretroviral effective amount.8. Methyl 6-deoxy-6-(N-acetamidino)-α-D-mannopyranoside.
 9. A conjugateof the formula:

wherein A is CH₃, X is a linear or branched C₁-C₈ alkyl chain optionallycontaining hydroxy, amino and/or oxo groups; n is an integer from 2 to6, and Sac is the residue of an aminoglycoside antibiotic.
 10. Aconjugate according to claim 9, wherein the aminoglycoside antibiotic isneomycin, kanamycin or gentamicin.
 11. A conjugate according to claim10, selected from the group consisting of the conjugatesγ-(N-acetamidino)butyric acid-neomycin B andtetra-γ-(N-acetamidino)butyric acid - kanamycin A.
 12. A conjugate ofthe formula:

wherein A is NH₂, X is a linear or branched C₁-C₈ alkyl chain optionallycontaining hydroxy, amino and/or oxo groups; n is an integer from 1 to6, and Sac is the residue of a mono- or oligo-saccharide, provided thatwhen X is —(CH₂)₃—CH(NH₂)—C(=O)—, the monosaccharide residue is notsubstituted at the position 1, and when Sac is the residue of anoligosaccharide, n is an integer from 2 to
 6. 13. A conjugate accordingto claim 12, wherein Sac is the residue of a monosaccharide.
 14. Aconjugate according to claim 13 selected from the group consisting ofthe conjugates methyl 6-deoxy-6-guanidino-α-D-mannopyranoside and methyl6-deoxy-6-(N-L-argininamido)-α-D-mannopyranoside.
 15. A conjugateaccording to claim 12, wherein Sac is the residue of an oligosaccharide.16. A conjugate according to claim 15, wherein Sac is the residue of anaminoglycoside antibiotic.
 17. A conjugate according to claim 16,wherein the aminoglycoside antibiotic is neomycin, kanamycin orgentamicin.
 18. The tetraargininamido-kanamycin A conjugate according toclaim 17, of the formula:


19. The triargininamido-gentamicin C conjugate according to claim 17, ofthe formula:


20. The tetraargininamido-gentamicin C conjugate according to claim 17,of the formula:


21. The hexa-argininamido-neomycin B conjugate according to claim 17, ofthe formula:


22. The γ-(N-guanidino)butyric acid-neomycin B conjugate according toclaim 17, of the formula:


23. The tetra-γ-(N-guanidino)butyric acid -kanamycin A conjugateaccording to claim 17, of the formula: